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In addition to rhizobia, many types of co-existent bacteria are found in leguminous root nodules, but their habitats are unclear. To investigate this phenomenon, we labeled Bradyrhizobium diazoefficiens USDA122 and Bradyrhizobium sp. SSBR45 with Discosoma sp. red fluorescent protein (DsRed) or enhanced green fluorescent protein (eGFP). USDA122 enhances soybean growth by forming effective root nodules, but SSBR45 does not form any nodules. Using low-magnification laser scanning confocal microscopy, we found that infected cells in the central zone of soybean nodules appeared to be occupied by USDA122. Notably, high-magnification microscopy after co-inoculation of non-fluorescent USDA122 and fluorescence-labeled SSBR45 also revealed that SSBR45 inhabits the intercellular spaces of healthy nodules. More unexpectedly, co-inoculation of eGFP-labeled USDA122 and DsRed-labeled SSBR45 (and vice versa) revealed the presence of USDA122 bacteria in both the symbiosomes of infected cells and in the apoplasts of healthy nodules. We then next inspected nodules formed after a mixed inoculation of differently-labeled USDA122, without SSBR45, and confirmed the inhabitation of the both populations of USDA122 in the intercellular spaces. In contrast, infected cells were occupied by single-labeled USDA122. We also observed Mesorhizobium loti in the intercellular spaces of active wild-type nodules of Lotus japonicus using transmission electron microscopy. Compatible intercellular rhizobia have been described during nodule formation of several legume species and in some mutants, but our evidence suggests that this type of colonization may occur much more commonly in leguminous root nodules.

We isolated a novel strain of Bradyrhizobium sp., SSBR45, from the nodulated roots of Aeschynomene indica and labeled it with Discosoma sp. red fluorescent protein (dsRED) or enhanced green fluorescent protein (eGFP) and determined its draft genomic sequence. The labeled SSBR45 stimulated the growth of A. indica markedly on a nitrogen-free medium, as observed by visualizing the fluorescent root nodules. The nodulated roots also exhibited high acetylene reduction activities. The SSBR45 genome included genes involved in nitrogen fixation, photosynthesis, and type IV secretion system; however, it did not consist of canonical nodABC genes and type III secretion system genes. SSBR45, a novel species of the genus Bradyrhizobium , consisted of an average nucleotide identity and average amino acid identity of 87% and 90%, respectively, with the closest strain B. oligotrophicum S58.

The circadian clock is synchronized by the day-night cycle to allow plants to anticipate daily environmental changes and to recognize annual changes in day length enabling seasonal flowering. This clock system has been extensively studied in Arabidopsis thaliana and was found to be reset by the dark to light transition at dawn. By contrast, studies on photoperiodic flowering of Pharbitis nil revealed the presence of a clock system reset by the transition from light to dark at dusk to measure the duration of the night. However, a Pharbitis photosynthetic gene was also shown to be insensitive to this dusk transition and to be set by dawn. Thus Pharbitis appeared to have two clock systems, one set by dusk that controls photoperiodic flowering and a second controlling photosynthetic gene expression similar to that of Arabidopsis. Here, we show that circadian mRNA expression of Pharbitis homologs of a series of Arabidopsis clock or clock-controlled genes are insensitive to the dusk transition. These data further define the presence in Pharbitis of a clock system that is analogous to the Arabidopsis system, which co-exists and functions with the dusk-set system dedicated to the control of photoperiodic flowering.

Purpose: To investigate the role of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in the medium-mediated bystander effect for chromosomal aberrations induced by low-linear energy transfer (LET) X-rays and high-LET heavy ions in normal human fibroblast cells., Materials and Methods: The recipient cells were treated for 12 h with conditioned medium, which was harvested from donor cells at 24 h after exposure to 10 Gy of soft X-rays (5 keV/microm) and 20Ne ions (437 keV/microm), followed by analyses of chromosome aberrations in recipient cells with premature chromosome condensation methods. To examine the role of DNA-PKcs and nitric oxide (NO), cells were treated with its inhibitor LY294002 (LY) and its scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (c-PTIO), respectively., Results: Increased frequency of chromosome aberrations in recipient cells treated with conditioned medium from irradiated but not from un-irradiated donor cells was observed which was independent of radiation type. Bystander induction of chromosome aberrations in recipient cells was mitigated when donor cells were treated with LY before irradiation and with c-PTIO after irradiation, and was enhanced when recipient cells were treated with LY before treatment of recipient cells with conditioned medium from irradiated donor cells., Conclusion: Irradiated normal human cells secrete NO and other molecules which in turn transmit radiation signals to unirradiated bystander cells, leading to the induction of bystander chromosome aberrations partially repairable by DNA-PKcs-mediated DNA damage repair machinery, such as non-homologous end-joining repair pathways.

Purpose: To quantify the Auger effect on chromosomal aberrations via Ca atoms in human cells., Material and Methods: Exponentially growing human normal fibroblasts (GM05389) were irradiated with 4.047 (CaK-P), 4.026 (CaK-L) and 4.067 (CaK-H) keV X-rays (corresponding to the resonance absorption edge of the Ca K-shell and slightly below and slightly above the edge, respectively) using synchrotron radiation at the photon factory (PF) of the High Energy Accelerator Organization located in Tsukuba. Chromosomal aberrations induced by the irradiation were analyzed by the premature chromosome condensation (PCC) method using calyculin A. The dependency of the chromosomal aberrations on the incubation time post 2 Gy irradiation was observed for each energy. Irradiation using 200 kVp conventional X-rays was also examined as a reference to CaK irradiation., Results: (1) Soon after irradiation with 2Gy, the enhancement ratios of CaK-H X-rays to CaK-L X-rays were 1.21, 1.51 and 2.70 for breaks/gaps, isochromatid breaks and exchanges, respectively. The enhancement ratios of CaK-P X-rays to CaK-L X-rays were 1.82, 0.98 and 6.30, for breaks/gaps, isochromatid breaks and exchanges, respectively. (2) After a 6-hr incubation treatment post 2 Gy irradiation, the enhancement ratios of CaK-H X-rays to CaK-L X-rays were 1.59, 2.03 and 2.14 for breaks/gaps, isochromatid breaks and exchanges, respectively. The enhancement ratios of CaK-P X-rays to CaK-L X-rays were 1.69, 1.66 and 2.00 for breaks/gaps, isochromatid breaks and exchanges, respectively. (3) Soon after irradiation, the ratios of the efficiencies of CaK-P X-rays to those of 200 kVp X-rays were 1.74, 1.29 and 2.51 for breaks/gaps, isochromatid breaks and exchanges, respectively. And after a 6-hr incubation treatment, the ratios were 5.50, 1.93 and 1.81 for breaks/gaps, isochromatid breaks and exchanges, respectively., Conclusions: An effective enhancement of chromosomal aberrations, such as breaks/gaps, isochromatid breaks and exchanges, was caused by Ca K-shell ionization or excitation. Auger electrons emitted by Ca atoms in irradiated cells appear to have an important role in causing this enhancement. Comparing these efficiencies of chromosomal aberrations with those produced by 200 kVp conventional X-rays suggests un-repaired and complicated damage is induced by the X-rays around the Ca K-shell resonance absorption edge.

The induction of single-strand breaks by vacuum-u.v. and far-u.v. radiations (150 nm-272 nm) in plasmid Col E1 DNA in buffered aqueous solution was studied by agarose gel electrophoresis. The results indicated that the mechanisms of DNA strand-break induction are different in the two u.v. regions: above 210 nm the direct excitation of DNA seemed to be the main cause; below 180 nm the photolysis of solvent water seemed to be essential. The demonstration of a systematic protection at 160 nm and not at 209 nm by OH scavengers, such as ethyl alcohol, potassium iodide and D-mannitol, supports the involvement of OH radicals in the vacuum-u.v. region.

The detailed binding mechanism of wheat germ agglutinin (WGA) with N-acetylglucosamine (GlcNAc) was investigated using intermolecular 1H-1H nuclear Overhauser effect (NOE) and atomic pair potential (APP) calculations. Negative NOE was observed on the 1H spectrum of 1-O-methyl derivative of GlcNAc in a solution containing WGA, when the aromatic region of the WGA spectrum was irradiated. Analyses of the time dependence of NOE revealed that H2 and the N-acetyl methyl protons of the sugar are in close proximity to the aromatic protons of WGA in the bound state. This was confirmed and further elucidated by the APP calculations. According to the calculation, the major binding force comes from a hydrogen-bonding between C3-OH of sugar and an acidic residue present in each of the two binding sites of WGA: Glu115 in site 1 and Asp29 in site 2. The binding is further assisted by the N-acetyl group which interacts with a few more polar amino acid residues in the binding sites. The optimized binding mode suggested by the APP calculations supports the NMR results in that H2 and a part of the N-acetyl methyl protons are within 4.5 A distance from protons of both Tyr64 and Tyr73 in site 1 and of Tyr159 in site 2.