Application of pseudomurein endoisopeptidase to fluorescence in situ hybridization of methanogens within the family Methanobacteriaceae

In situ detection of methanogens within the family Methanobacteriaceae is sometimes known to be unsuccessful due to the difficulty in permeability of oligonucleotide probes. Pseudomurein endoisopeptidase (Pei), a lytic enzyme that specifically acts on their cell walls, was applied prior to 16S rRNA-targeting fluorescence in situ hybridization (FISH). For this purpose, pure cultured methanogens within this family, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, and Methanothermobacter thermautotrophicus together with a Methanothermobacter thermautotrophicus-containing syntrophic acetate-oxidizing coculture, endosymbiotic Methanobrevibacter methanogens within an anaerobic ciliate, and an upflow anaerobic sludge blanket (UASB) granule were examined. Even without the Pei treatment, Methanobacterium bryantii and Methanothermobacter thermautotrophicus cells are relatively well hybridized with oligonucleotide probes. However, almost none of the cells of Methanobrevibacter ruminantium, Methanosphaera stadtmanae, cocultured Methanothermobacter thermautotrophicus, and the endosymbiotic methanogens and the cells within UASB granule were hybridized. Pei treatment was able to increase the probe hybridization ratio in every specimen, particularly in the specimen that had shown little hybridization. Interestingly, the hybridizing signal intensity of Methanothermobacter thermautotrophicus cells in coculture with an acetate-oxidizing H(2)-producing syntroph was significantly improved by Pei pretreatment, whereas the probe was well hybridized with the cells of pure culture of the same strain. We found that the difference is attributed to the differences in cell wall thicknesses between the two culture conditions. These results indicate that Pei treatment is effective for FISH analysis of methanogens that show impermeability to the probe.     

Termite-regulated fungal monoculture in fungus combs of a macrotermitine termite Odontotermes formosanus

The mechanism of the exclusive growth of Termitomyces in fungus combs with fungi-growing termites, O. formosanus was examined using laboratory scale fungus combs. In the combs without the termites, vigorous growth of unidentified fungi was observed although no significant change was found in the case of the combs with termites. In addition, these results were reproducible even when incubated in a separated dish, suggesting that the physicochemical conditions were not the reason for the growth. With the molecular based analysis for the microbial communities in the combs, monoculture of the Termitomyces in the combs with termites was confirmed while the bacterial communities were independent either with or without termites. Possible mechanism of the exclusive growth of Termitomyces, such as the selective grazing of pathogenic fungi or contribution of antifungal activity giving actinomycetes were also discussed.     

Influence of nitrogen fertilization on tropical‐grass silage assessed by ensiling process monitoring using chemical and microbial community analyses

Aims: Utilization of silage in livestock farming is expected to increase in developing countries in the tropical and subtropical parts of the world. The aim of this study was to investigate the influence of nitrogen fertilization on the chemical composition of herbage, ensiling process and silage quality, and to contribute to the improvement of tropical‐grass silage preparation. Methods and Results: Guinea grass grown under two different nitrogen‐fertilizer application conditions [1·5 kg N a^?1 (high‐N) and 0·5 kg N a^?1 (low‐N)] was packed in plastic bags, and its ensiling process was investigated by chemical and microbial‐community analyses. Relatively well‐preserved silage was obtained from high‐N herbage, which accumulated a high nitrate concentration. Denaturing gradient gel electrophoresis analysis revealed that Lactobacillus plantarum dominated throughout the ensiling of high‐N herbage and in the early phase of that of low‐N herbage. In low‐N silages prepared from ammonium sulfate‐ and urea‐fertilized herbage, Lact. plantarum was replaced by clostridia after 40 and 15 days of ensiling, respectively. Conclusions: Nitrate content of herbage is an important factor that influences silage quality, and careful fertilization management can facilitate stable and successful fermentation of tropical‐grass silage without any pretreatment. Significance and Impact of the Study: The positive effect of nitrate on the ensiling process of tropical‐grass was proved by microbial‐community analysis.     

5-O-glucosyldihydroflavones from the leaves of Helicia cochinchinensis

From the leaves of Helicia cochinchinensis, collected on Okinawa Island, seven phenolic glucosides and two terpenic glucosides were isolated. Five of the phenolic glucosides were previously known, being identified with p-coumaric and ferulic acids glucosyl esters, rhodioloside, helicidiol, and naringenin 5-O-beta-D-glucopyranoside. The structures of two other phenolic glucosides, named heliciosides A and B, were elucidated to be 5-O-beta-D-glucosides of 3-hydroxyflavanone, namely aromadendrin and taxifolin, by means of spectroscopic analyses. The two terpenic glucosides were identified with ampelopsisionoside and icariside C1.     

Identification of fast-evolving genes in the scleractinian coral Acropora using comparative EST analysis

To identify fast-evolving genes in reef-building corals, we performed direct comparative sequence analysis with expressed sequence tag (EST) datasets from two acroporid species: Acropora palmata from the Caribbean Sea and A. millepora from the Great Barrier Reef in Australia. Comparison of 589 independent sequences from 1,421 A. palmata contigs, with 10,247 A. millepora contigs resulted in the identification of 196 putative homologues. Most of the homologous pairs demonstrated high amino acid similarities (over 90%). Comparisons of putative homologues showing low amino acid similarities (under 90%) among the Acropora species to the near complete datasets from two other cnidarians (Hydra magnipapillata and Nematostella vectensis) implied that some were non-orthologous. Within 86 homologous pairs, 39 exhibited dN/dS ratios significantly less than 1, suggesting that these genes are under purifying selection associated with functional constraints. Eight independent genes showed dN/dS ratios exceeding 1, while three deviated significantly from 1, suggesting that these genes may play important roles in the adaptive evolution of Acropora. Our results also indicated that CEL-III lectin was under positive selection, consistent with a possible role in immunity or symbiont recognition. Further studies are needed to clarify the possible functions of the genes under positive selection to provide insight into the evolutionary process of corals.     

Disturbance response indicators of Impatiens walleriana exposed to benzene and chromium

The purpose of this study was to evaluate the remediation potential and disturbance response indicators of Impatiens walleriana exposed to benzene and chromium. Numerous studies over the years have found abundant evidence of the carcinogenicity of benzene and chromium (VI) in humans. Benzene and chromium are two toxic industrial chemicals commonly found together at contaminated sites, and one of the most common management strategies employed in the recovery of sites contaminated by petroleum products and trace metals is in situ remediation. Given that increasing interest has focused on the use of plants as depollution agents, direct injection tests and benzene misting were performed on I. walleriana to evaluate the remediation potential of this species. I. walleriana accumulated hexavalent chromium, mainly in the root system (164.23 mg kg^?1), to the detriment of the aerial part (39.72 mg kg^?1), and presented visible damage only at the highest concentration (30 mg L^?1). Unlike chromium (VI), chromium (III) was retained almost entirely by the soil, leaving it available for removal by phytotechnology. However, after the contamination stopped, I. walleriana responded positively to the detoxification process, recovering its stem stiffness and leaf color. I. walleriana showed visible changes such as leaf chlorosis during the ten days of benzene contamination. When benzene is absorbed by the roots, it is translocated to and accumulated in the plant's aerial part. This mechanism the plant uses ensures its tolerance to the organic compound, enabling the species to survive and reproduce after treatment with benzene. Although I. walleriana accumulates minor amounts of hexavalent chromium in the aerial part, this amount suffices to induce greater oxidative stress and to increase the amount of hydrogen peroxide when compared to that of benzene. It was therefore concluded that I. walleriana is a species that possesses desirable characteristics for phytotechnology.     

High cell density cultivation of recombinant Escherichia coli for hirudin variant 1 production

A synthetic medium, TK-25, for high cell density cultivation (HCDC) of Escherichia coli K-12 was modified to support HCDC of strain JM109. By optimizing the culture conditions, the cell concentration of 65 g/l in 14 h was obtained in the optimized medium, namely TK-10, with glucose-fed batch cultivation. When these conditions were further applied for HCDC of E. coli JM109 harboring pUC-based recombinant plasmid which expresses a hirudin variant, HV-1-fused protein under the control of trp promoter, it grew to 24 g/l of dried cells expressed as an inclusion body as 15.9% of the total protein, corresponding to 1908 mg/l hirudin-fused protein.     

Chlorine-containing iridoid and iridoid glucoside, and other glucosides from leaves of Myoporum bontioides

Sixteen compounds were isolated from the MeOH extract of leaves of Myoporum bontioides. The five compounds hitherto unknown, were elucidated to be a chlorine-containing iridoid, named myopochlorin, and an iridoid glucoside, an acylated iridoid glucoside, a linear acetogenin glucoside, and an acyclic monoterpene glucoside, named myobontiosides A-D, respectively, by means of spectroscopic analyses.