Akiyo Yamada

Microaerobic hydrogen production by photosynthetic bacteria in a double-phase photobioreactor

The rate of hydrogen production by the marine nonsulfur photosynthetic bacterium, Rhodovulum sp., increased with increasing light intensity. A light intensity of 1800 W/m(2) hydrogen production rate was achieved at the rate of 9.4 micromol/mg dry weight/h. The hydrogen production of this strain was enhanced by the addition of a small amount of oxygen (12 micromol O(2)/reactor). Intracellular ATP content was most efficiently accumulated under microaerobic, dark conditions. Hydrogen production rate by Rhodovulum sp. was investigated using a double-phase photobioreactor consisting of light and dark compartments. This rate was compared with data obtained using a conventional photobioreactor. Rhodovulum sp. produced hydrogen at a rate of 0.38+/-0.03 micromol/mg dry weight/h under microaerobic conditions using the double-phase photobioreactor. The hydrogen production rate was four times greater under microaerobic conditions, as compared with anaerobic conditions using either type of photobioreactor. Hydrogen production using a double-phase photobioreactor was demonstrated continuously at the same rate for 150 h.

Extracellular reduction of selenite by a novel marine photosynthetic bacterium.

Abstract A novel purple nonsulfur bacterium strain NKPB030619, which has resistance to over 5 mM selenite, was isolated from a marine environment. An initial concentration of 1.1 mM selenite, added to the medium, was decreased to under 0.05 mM within 5 days. The color of the cell suspension turned red within 2 days. The red coloration gradually decreased and black precipitates appeared during 2 weeks of cultivation. Under these conditions, two main types of deposit were formed extracellularly. These deposits were thought to contain red amorphous selenium and black vitreous selenium. The selenite reduction to elemental selenium in this bacterium was induced by the introduction of light and l-malic acid under anaerobic conditions. These results suggest that selenite reduction is coupled with photosynthesis and l-malic acid can serve as the indirect electron donor for its reduction. Phylogenetic analysis based on the 16S rDNA sequence showed that NKPB0360619 belongs to the α subdivision of Proteobacteria and is classified into the Rhodobacter species. The highest similarity of 86.2% was observed with R. sphaeroides.

Assessment of the salt tolerance and environmental biosafety of Eucalyptus camaldulensis harboring a mangrin transgene.

Increasing soil salinization of arable land has a major impact on the global ecosystem. One approach to increase the usable global forest area is to develop transgenic trees with higher tolerance to conditions of salt stress. An allene oxide cyclase homolog, mangrin, contains a core protein domain that enhances the salt tolerance of its host. We utilized this feature to develop improved salt-tolerant eucalyptus trees, by using transgenic Eucalyptus camaldulensis carrying the mangrin gene as a model. Since the Japanese government requires an environmental biosafety assessment for the surrounding biosphere, we performed experiments on trees grown in a special netted-house. This study examined the transgenic E. camaldulensis carrying the mangrin gene to assess the feasibility of using these transformants, and assessed their salt tolerance and environmental biosafety. We found that seven of 36 transgenic genotypes had significantly higher salt tolerance than non-transformants, and more importantly, that these plants had no significant impact on environmental biosafety. These results suggest that introduction of the mangrin gene may be one approach to safely enhance salt tolerance in genetically modified Eucalyptus species, and that the transformants have no apparent risks in terms of environmental biosafety. Thus, this study provides valuable information regarding the use of transgenic trees in situ.

Differential Analysis of Protein Expression in RNA-Binding-Protein Transgenic and Parental Rice Seeds Cultivated under Salt Stress

Transgenic plants tolerant to various environmental stresses are being developed to ensure a consistent food supply. We used a transgenic rice cultivar with high saline tolerance by introducing an RNA-binding protein (RBP) from the ice plant (Mesembryanthemum crystallinum); differences in salt-soluble protein expression between nontransgenic (NT) and RBP rice seeds were analyzed by 2D difference gel electrophoresis (2D-DIGE), a gel-based proteomic method. To identify RBP-related changes in protein expression under salt stress, NT and RBP rice were cultured with or without 200 mM sodium chloride. Only two protein spots differed between NT and RBP rice seeds cultured under normal conditions, one of which was identified as a putative abscisic acid-induced protein. In NT rice seeds, 91 spots significantly differed between normal and salt-stress conditions. Two allergenic proteins of NT rice seeds, RAG1 and RAG2, were induced by high salt. In contrast, RBP rice seeds yielded seven spots and no allergen spots with significant differences in protein expression between normal and salt-stress conditions. Therefore, expression of fewer proteins was altered in RBP rice seeds by high salt than those in NT rice seeds.

Phosphorus accumulation by a marine photosynthetic bacterium, Chromatium sp.

Among 8 strains of typical marine and freshwater photosynthetic bacteria, a Chromatium sp., which is known to flocculate upon the addition of seawater, had the highest intracellular phosphorus content of 0.865 mmol P/mg dry wt with a ratio of polyphosphate to phosphate (poly-Pi/Pi) of 2.14. Intracellular phosphorus content increased withincreasing NaCl concentration up to 260 mM. The poly-Pi/Pi value was also the highest in the medium containing 260 mM NaCl. Sodium cation in seawater enhanced intracellular phosphorus accumulation in this bacterium.

Heterologous expression of a plant RelA-SpoT homologue results in increased stress tolerance in Saccharomyces cerevisiae by accumulation of the bacterial alarmone ppGpp

The bacterial alarmone ppGpp is present only in bacteria and the chloroplasts of plants, but not in mammalian cells or eukaryotic micro-organisms such as yeasts and fungi. The importance of the ppGpp signalling system in eukaryotes has therefore been largely overlooked. Here, we demonstrated that heterologous expression of a relA-spoT homologue (Sj-RSH) isolated from the halophilic plant Suaeda japonica in the yeast Saccharomyces cerevisiae results in accumulation of ppGpp, accompanied by enhancement of tolerance against various stress stimuli, such as osmotic stress, ethanol, hydrogen peroxide, high temperature and freezing. Unlike bacterial ppGpp accumulation, ppGpp was accumulated in the early growth phase but not in the late growth phase. Moreover, nutritional downshift resulted in a decrease in ppGpp level, suggesting that the observed Sj-RSH activity to synthesize ppGpp is not starvation-dependent, contrary to our expectations based on bacteria. Accumulated ppGpp was found to be present solely in the cytosolic fraction and not in the mitochondrial fraction, perhaps reflecting the ribosome-independent ppGpp synthesis in S. cerevisiae cells. Unlike bacterial inosine monophosphate (IMP) dehydrogenases, the IMP dehydrogenase of S. cerevisiae was insensitive to ppGpp. Microarray analysis showed that ppGpp accumulation gave rise to marked changes in gene expression, with both upregulation and downregulation, including changes in mitochondrial gene expression. The most prominent upregulation (38-fold) was detected in the hypothetical gene YBR072C-A of unknown function, followed by many other known stress-responsive genes. S. cerevisiae may provide new opportunities to uncover and analyse the ppGpp signalling system in eukaryotic cells.

Putative cis-elements in the promoter region of the carrot phenylalanine ammonia-lyase gene induced during anthocyanin synthesis

Deletion mutants of the carrot phenylalanine ammonia-lyase gene promoter were used to survey cis-elements for their effect on expression of promoter activity by transient expression. Two putative cis-elements were required to give full activity, but a third might be the most important in regulation of the promoter by 2,4-dichlorophenoxyacetic acid.

Development of a phosphate-removal system using a marine photosynthetic bacterium Chromatium sp.

Abstract The marine photosynthetic bacterium Chromatium sp. successfully removed orthophosphate when grown phototrophically. The phosphate-uptake rate was almost constant at more than 5.0 mg- PO43−/l in synthetic medium. Addition of seawater causes flocculation of this strain. The successful use of seawater as an inexpensive source of magnesium could prove to be effective in the removal of photosynthetic bacterial cells from a medium. A semicontinuous culture system was used for the removal of low concentrations of phosphate and the phosphate-uptake activity of Chromatium sp. was maintained under 0.1 day−1 dilution rate. This strain was also able to remove high concentrations of phosphate from domestic sewage.

Development and evaluation of novel salt-tolerant Eucalyptus trees by molecular breeding using an RNA-Binding-Protein gene derived from common ice plant (Mesembryanthemum crystallinum L.)

Summary The breeding of plantation forestry trees for the possible afforestation of marginal land would be one approach to addressing global warming issues. Here, we developed novel transgenic Eucalyptus trees (Eucalyptus camaldulensis Dehnh.) harbouring an RNA‐Binding‐Protein (McRBP) gene derived from a halophyte plant, common ice plant (Mesembryanthemum crystallinum L.). We conducted screened‐house trials of the transgenic Eucalyptus using two different stringency salinity stress conditions to evaluate the plants’ acute and chronic salt stress tolerances. Treatment with 400 mM NaCl, as the high‐stringency salinity stress, resulted in soil electrical conductivity (EC) levels >20 mS/cm within 4 weeks. With the 400 mM NaCl treatment, >70% of the transgenic plants were intact, whereas >40% of the non‐transgenic plants were withered. Treatment with 70 mM NaCl, as the moderate‐stringency salinity stress, resulted in soil EC levels of approx. 9 mS/cm after 2 months, and these salinity levels were maintained for the next 4 months. All plants regardless of transgenic or non‐transgenic status survived the 70 mM NaCl treatment, but after 6‐month treatment the transgenic plants showed significantly higher growth and quantum yield of photosynthesis levels compared to the non‐transgenic plants. In addition, the salt accumulation in the leaves of the transgenic plants was 30% lower than that of non‐transgenic plants after 15‐week moderate salt stress treatment. There results suggest that McRBP expression in the transgenic Eucalyptus enhances their salt tolerance both acutely and chronically.