• Journal of Microbiology and Biotechnology
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• v.25 no.8
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• pp.1339-1348
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• 2015

Until now, considerable effort has been made to engineer novel nitrogen-fixing organisms through the transfer of nif genes from various diazotrophs to non-nitrogen fixers; however, regulatory coupling of the heterologous nif genes with the regulatory system of the new host is still not well understood. In this work, a 49 kb nitrogen fixation island from P. stutzeri A1501 was transferred into E. coli using a novel and efficient transformation strategy, and a series of recombinant nitrogen-fixing E. coli strains were obtained. We found that the nitrogenase activity of the recombinant E. coli strain EN-01, similar to the parent strain P. stutzeri A1501, was dependent on external ammonia concentration, oxygen tension, and temperature. We further found that there existed a regulatory coupling between the E. coli general nitrogen regulatory system and the heterologous P. stutzeri nif island in the recombinant E. coli strain. We also provided evidence that the E. coli general nitrogen regulator GlnG protein was involved in the activation of the nif-specific regulator NifA via a direct interaction with the NifA promoter. To the best of our knowledge, this work plays a groundbreaking role in increasing understanding of the regulatory coupling of the heterologous nitrogen fixation system with the regulatory system of the recipient host. Furthermore, it will shed light on the structure and functional integrity of the nif island and will be useful for the construction of novel and more robust nitrogen-fixing organisms through biosynthetic engineering.

• The Korean Journal of Ecology
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• v.18 no.1
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• pp.43-54
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• 1995

Effects of environmental factors of N, P, pH, moisture, temperature and oxygen on growth and nitrogen fixation activity of kummerowia striate (Thunb.) Schindler seedling, bearing symbiotic root nodules, were quantitatively analyzed during the growing period. The specific nitrogenase activity (ARA) of nodules showed the maximum value of 187 μmol C₂H₄g fr wt-1 h-1 6 weeks after seeds were germinated. The total nitrogenase activities per plant attained as 1.56, 0.85, 0.09 and 4.0, 1.11, 0.04 μmol C₂H₄hr-1, respectively for the treatments of 1, 3 and 5 mM NO₃￣and NH₄＋ on the 60th day. While the plant grown in N-free media for 20 days after treatments of 5 mM NH₄+for 40 days resulted in 30 mg fr wt of nodule formation and exhibited the relative activities of 152％ and 162％ for total and specific ARA in comparison with those of control plant grown with N-free for 60 days. Total biomass and ARA was by 70％ and 86％ lower in N and P deficiency, respectively. The N and P deficient plot showed 70％ and 86％ decreases of total biomass and ARA in comparison with those of control. The plant grown with N-free for 20 days after pretreatment with N and P free media for 40 days showed the relative values of 77％, 118％ and 150％, respectively for nodule biomass, total and specific ARA in comparison with those of control. The treatment with acid or alkali gradients resulted in significant decreases of nodule biomass and ARA. The optimum temperature and pO₂for ARA were 30°C and 40 kPa, respectively. Two peaks of diurnal variation appeared at 11:00 and 23:00 o'clocks by the continuous light condition. The plants with water stress by temporary wilting point rsulted in 95~97％ inhibition for nodule respiration, transpiration and specific ARA. Transpiration and ARA ware recovered to 88％ and 38％ of those of water unstressed plants, respectively, 6 hours after the plants were rewatered from water stressed condition.

• The Korean Journal of Ecology
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• v.21 no.3
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• pp.179-186
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• 1998

Effects of lead(Pb) and calcium(Ca) on growth responses, accumulation of heavy metals and nitrogen fixation activities of Melilotus suaveolens seedlings were quantitatively analyzed during growing period. Pb contents of the root treated with 30, 100 ppm Pb and 100 ppm Pb added 100 ppm Ca were 54.1, 90.9 and 26.1 folds higher than that of the control, respectively, at pH 4.2 in 28th days, and heavy metal content of plant increased with increasing of pH and Pb concentration. The melilot plant was classified as a Pb accumulator by higher accumulation of Pb in shoot than that of root. Pb treatments resulted in inhibiton of height and chlorophyll contet, and Ca treatment increased height and chlorophyll content insignificantly at pH 4.2 in 28 days. The plant biomass reduced 49, 60 and 54% at pH 4.2 and 47,53 and 50% at pH 6.5, respectively, by 30, 100 ppm Pb and 100 ppm Pb added 100 ppm Ca treatment. Specific nitrogen fixation of nodules reduced 68.4% and 46.6% by 100 ppm Pb treatment and 3.7% and 24.9% by 100 ppm Pb added 100 ppm Ca at pH 4.2 and pH 6.5, respectively, so Ca inhibited significantly Pb activity and toxicity in acdic pH. Nodule formation were reduced to 33, 33 and 50% at pH 4.2 and 50, 33 and 38% at pH 6.5 by 30, 100 ppm Pb and 100 ppm Pb added 100 ppm Ca, respectively.

• Journal of Ecology and Environment
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• v.34 no.3
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• pp.279-286
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• 2011

The effect of allelochemicals on growth, root nodule nitrogen fixation activity, and ion patterns of soybeans were investigated. We prepared 50 g/L (T50), 100 g/L (T100), and 200 g/L (T200) extract concentrations by soaking fresh red pine needles in a nutrient solution. Adding needles to the nutrient solution increased the content of total phenolic acids, osmolality, and total ions. The total phenolic content in the T50, T100, and T200 extracts were $206{\pm}12.61$, $335{\pm}24.16$, and $603{\pm}12.30$ mg gallic acid equivalents, respectively. The $K^+$, $Mg^{2+}$, $Ca^{2+}$, and $PO_4^{3-}$ content increased by adding needles to the nutrient solutions, whereas $SO_4^{2-}$ content decreased. The growth inhibition of soybeans was proportional to the needle extract concentrations, and the T100 and T200 concentrations resulted in remarkable growth inhibition. On day 20 after treatment, dry weight and nitrogen fixation activity of the root nodules were reduced by the T100 and T200 treatments, whereas the T50 treatment was not difference from the control. After day 10, total ion content in all treatment groups was not different in comparison with the control. However, total ionic content in all treatment groups decreased significantly compared with that in the control after day 20. The lowest total ion value was found for the T200 concentration. The T200 treatment also resulted in significantly reduced $SO_4^{2-}$ content. The amounts of $Mg^{2+}$, $Ca^{2+}$, and $Mn^{2+}$ were higher than those of the control for the T50 treatment on day 10 and for T100 on day 20 after treatment. A significant increase in osmolality was observed in the T200 treatment on day 10 and in the T100 treatment on day 20. These results suggest that under severe allelochemical stress conditions, a remarkable reduction in nodule formation, nitrogen fixation activity, and ion uptake eventually resulted in a decrease in leaf production. Furthermore, increased $K^+$, $Mg^{2+}$, $Ca^{2+}$, $Mn^{2+}$, and osmolality in soybeans exposed to lower concentrations of allelochemicals than the critical stress level helped overcome the stress.

• Journal of The Korean Society of Grassland and Forage Science
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• v.14 no.2
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• pp.88-92
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• 1994

This study was carried out to investigate the effects of lime(0, 250, 500 and 1,000 kg/lOa) and phosphate (0, 17 and 34 kg/lOa) applications on growth and nitrogen fixation of alfalfa (Medicago sativa L.). The phosphate applications improved(p<0.05) shoot, root and root nodule dry weight of Alfalfa and acetylene reduction activity of alfalfa with increasing levels of phosphate in the soil pH 6.2 at 7 and 12 weeks(ear1y bloom) after sowing, but these were not influnced with lime applications. Total nitrogen content of each part of alfalfa was increased with lime application, but was not significantly different by levels of lime application at 7 weeks after sowing. Application of phosphate did not affect total nitrogen content of each part of alfalfa. Total nitrogen contents of each part of alfalfa were not significantly different between lime and non-lime application at 12 weeks(ear1y bloom) after sowing but were decreased(p<0.05) with phosphate application(p< 0.05). These results suggest that lime(250 kg/lOa) and phosphate(34 kg/lOa) applications are effective for the growth and the nitrogen fixation of alfalfa at the soil pH 6.20.

• The Korean Journal of Food And Nutrition
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• v.5 no.2
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• pp.137-143
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• 1992

As a research for developing biofertilizers, Klebsiella oxytoca, an associative nitrogen fixer in the rhizosphere of rice plant in the soil of paddy field, was subjected to molecular breeding. The results obtained were as followings. 1). By transforming pbIC71A, Nif A overproducing plasmid, into Klebsiella oxytoca NGl3, Klebsiell6f oxytoca SH3l, and Klebsiella oxytoca SH161, nitrogenase activities in the absence of nitrogen source in the medium were increased 6.4, 17.2, and 13.5 times, respectively, in comparison with the parent strains. 2). Nitrogenase activity of Klebgiella oxytoca NGl3, Klebsiella oxytoca SH3l, and Klebsiella oxytoca SH161 was completely repressed In the presence of 15mM NH4+. But, nitrogenase activities of Klebsiella oxytoca NGl3/PMC71A, Klebsiella oxytoca SH3l /PMC71A, and Klebsiella oxytoca SH 161/pMC714 harboring PMC71A, were 13.7%, 7.7%, and 6.2% of the nitrogenase activities in the absence of nitrogen source in the medium, respectively.

• Journal of Plant Biology
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• v.29 no.1
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• pp.67-75
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• 1986

An experimental system for the possibility of association between cultured plant cells and a cyanobacterium (N. muscorum) was investigated. The association was established in nitrogen-free medium, and nitrogen source was provided by N. muscorum through its own nitrogen fixation. N. muscorum was distributed on the outer part and intercellular spaces of the cultured plant cells. Polyamines were treated to the nitrogen-free medium to improve the association. Polyamines increased nitrogenase activity and protein amount in the association between N. muscorum and the cultured plant cells, as compared to the association without polyamines. Especially, spermine showed an increasing effect on the nitrogenase activity and the protein content in the association of tobacco and N. muscorum, while spermidine showed similar effect in case of soybean and N. muscorum.

• Journal of Plant Biology
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• v.35 no.4
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• pp.317-322
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• 1992

The nitrogen fixation activity and environmental factors of Lespedeza bicli!oy Turcz, forming annual root nodules by symbiotic Rhizobium sp. were analyzed in the field conditions during the growing period. Seasunal changes of $N_2-fixation$ activity showed the maximum value of $120\;\mu\textrm{M}\;C_2_H4{\cdot}noduie\;g\;fw^{-1}{\cdot}hr^{-1}$ during the active growing period (June) and varied significantly depending on the growth phase and environmental factors. The maximum activities were attained at the conditions of pH 7, $30^{\circ}C$ of temperature, 18 Kpa of oxygen partial pressure and inhibited by water stress and nitrogen sources. The habitat soil was weak acidic and poor in nitrogen, phosphorus and organic matter contents. The leaf area ratios and chlorophyll contents were ranged from 442 to $48;\textrm{cm}^2{\cdot}g\;dw^{-1}$ and from 33 to $38\;\mu\textrm{g}\;chI{\cdot}\textrm{cm}^2$. Nitrogen contents in each organ showed the maximum of 46, 19 and $11\;mg{\cdot}g\;dw^{-1}, respectively for leaf, rool and stem in the early period. The highest phosphorus contents were 4.2, 1.2 and $0.6\;mg{\cdot}g\;dw^{-1}$, respectively for leaf. root and stem in early growing period. The allocation ratios of nitrogen quantity showed 60% for leaves and 73% for roots during the active and late growth period, and 22% [or stems in average. The allocalion ralios o[ phosphorus quantity showed 58% for leaves during the most productive period, 70% for roots in the pre-growth stage and 26% for stems in average.verage.

• Journal of Microbiology and Biotechnology
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• v.12 no.3
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• pp.423-430
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• 2002

The effects of UV-B and commercial grade pesticides (butachlor and carbofuran), individually and in combination, were studied on a variety of physiological processes of rice field cyanobacterium Anabaena doliolum. Butachlor was found to be $2-12\%$ more toxic than carbofuran and $4-24\%$ than UV-B on the growth, photosynthesis, lipid peroxidation, membrane permeability, and nitrogenase activity of the test cyanobacterium. Of the three photosynthesis inhibitors, the butachlor-induced inhibition of whole chain was approximately 3 and $21\%$ higher than carbofuran and UV-B, respectively. Although the interaction of the stress factors caused a significant inhibition (P<0.01), it was still less than the additive effect on the parameters investigated, except for PSI.

• Proceedings of the Korean Society of Crop Science Conference
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• 1986.06a
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• pp.64-65
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• 1986