Identification of a 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid Reductase,FlRed, in an Alginolytic Bacterium Flavobacterium sp. Strain UMI-01

In alginate-assimilating bacteria, alginate is depolymerized to unsaturated monosaccharide by the actions of endolytic and exolytic alginate lyases (EC 4.2.2.3 and EC 4.2.2.11). The monosaccharide is non-enzymatically converted to 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH), then reduced to 2-keto-3-deoxy-d-gluconate (KDG) by a specific reductase, and metabolized through the Entner–Doudoroff pathway. Recently, the NADPH-dependent reductase A1-R that belongs to short-chain dehydrogenases/reductases (SDR) superfamily was identified as the DEH-reductase in Sphingomonas sp. A1. We have subsequently noticed that an SDR-like enzyme gene, flred, occurred in the genome of an alginolytic bacterium Flavobacterium sp. strain UMI-01. In the present study, we report on the deduced amino-acid sequence of flred and DEH-reducing activity of recombinant FlRed. The deduced amino-acid sequence of flred comprised 254 residues and showed 34% amino-acid identities to that of A1-R from Sphingomonas sp. A1 and 80%–88% to those of SDR-like enzymes from several alginolytic bacteria. Common sequence motifs of SDR-superfamily enzymes, e.g., the catalytic tetrad Asn-Lys-Tyr-Ser and the cofactor-binding sequence Thr-Gly-x-x-x-Gly-x-Gly in Rossmann fold, were completely conserved in FlRed. On the other hand, an Arg residue that determined the NADPH-specificity of Sphingomonas A1-R was replaced by Glu in FlRed. Thus, we investigated cofactor-preference of FlRed using a recombinant enzyme. As a result, the recombinant FlRed (recFlRed) was found to show high specificity to NADH. recFlRed exhibited practically no activity toward variety of aldehyde, ketone, keto ester, keto acid and aldose substrates except for DEH. On the basis of these results, we conclude that FlRed is the NADH-dependent DEH-specific SDR of Flavobacterium sp. strain UMI-01.     

Entry Inhibition of Influenza Viruses with High Mannose Binding Lectin ESA-2 from the Red Alga Eucheuma serra through the Recognition of Viral Hemagglutinin

Lectin sensitivity of the recent pandemic influenza A virus (H1N1-2009) was screened for 12 lectins with various carbohydrate specificity by a neutral red dye uptake assay with MDCK cells. Among them, a high mannose (HM)-binding anti-HIV lectin, ESA-2 from the red alga Eucheuma serra, showed the highest inhibition against infection with an EC₅₀ of 12.4 nM. Moreover, ESA-2 exhibited a wide range of antiviral spectrum against various influenza strains with EC₅₀s of pico molar to low nanomolar levels. Besides ESA-2, HM-binding plant lectin ConA, fucose-binding lectins such as fungal AOL from Aspergillus oryzae and AAL from Aleuria aurantia were active against H1N1-2009, but the potency of inhibition was of less magnitude compared with ESA-2. Direct interaction between ESA-2 and a viral envelope glycoprotein, hemagglutinin (HA), was demonstrated by ELISA assay. This interaction was effectively suppressed by glycoproteins bearing HM-glycans, indicating that ESA-2 binds to the HA of influenza virus through HM-glycans. Upon treatment with ESA-2, no viral antigens were detected in the host cells, indicating that ESA-2 inhibited the initial steps of virus entry into the cells. ESA-2 would thus be useful as a novel microbicide to prevent penetration of viruses such as HIV and influenza viruses to the host cells.     

海岸湿地针叶林-阔叶林突变临界维持机制研究

日本北海道东部有两块发展成熟的湿地森林,分别是具有氮固定作用的日本桤木（Alnusjaponica Steud）组成的阔叶林和库页云杉（Piceaglehnii Masters）构成的针叶林,而且两块林地间具有突变的边界。为了阐明突变边界的维持机制,本文研究了森林剖面、水位、地下水、降雨化学、与微环境有关的幼苗建成格局及种子迁移。研究表明,地下水位剖面并不能充分地解释突转临界的形成,但是,两种林地的地下水化学成分存在显著差异,具体表现为库页云杉林地下水中EC,Na^＋,心,Mg^2＋,Ca^2＋和Cl^-含量明显要高于日本桤木林的,而地下水pH值又较低。库页云杉林降雨中富含Na^＋,Ca^2＋和Cl^-,表明降雨是导致两种林分土壤表层水化学成分差异的主要原因。六月末,库页云杉林的太阳辐射少于2．2MJ·m^-2．d^-1;而日本桤木林太阳辐射强度为1．0—3．7MJ·m^-2.d^-1。土壤表层苔藓主要是泥炭藓,在库页云杉林内覆盖率为60％,而日本桤木林的仅为10％：这一差异与土壤表层水pH值有关。有70％株高在1．3米以下的库页云杉苗木建立在苔藓层上。播种造林实验表明,在库页云杉林2个树种的种子萌发率和幼苗存活率都较高。因此,库页云杉在日本桤木林内繁殖能力较弱,主要是缺少适宜的微环境和较低的苗木建成能力。日本桤木仅靠再萌发繁殖,在两种林分内幼苗较少;此外,日本桤木种子向库页云杉林内的迁移也受到较大的限制,加之库页云杉林内太阳辐射强度较低,导致日本桤木幼苗存活率较低。基于以上结果,我们认为日本桤木与库页云杉形成了各自特有的生长环境,而对彼此的生长环境不适应,抑制了自身种苗向对方林分的扩张和自然再造过程。图9表1参32。     

Species diversity of the marine diatom genus Skeletonema in Japanese brackish water areas

The genus Skeletonema includes phytoplankton species that are important primary producers in marine food chains. Brackish waters have been reported to be one of the important habitats of some species of Skeletonema. To elucidate the species diversity of Skeletonema in brackish waters, we investigated three Japanese brackish bodies of water: the coastal waters of Toyama Bay, a tidal area of the Chikugo River, and a constructed reservoir in Isahaya Bay. We used molecular analysis based on large subunit rDNA and fine morphological structure to identify species. Skeletonema costatum s.s. (sensu stricto) was isolated at salinities as low as 0.6, but Skeletonema dohrnii, Skeletonema subsalsum, and Skeletonema tropicum were not found at salinities below 11.0. S. costatum s.s. could survive transfer from a medium with a salinity of 15 to a salinity of 2, but S. dohrnii did not survive in the same experiment. Only S. costatum s.s. germinated from the sediment of a reservoir in which the salinity was 0.1–1.4; incubation conditions included temperatures of 10, 15, 20, 25, and 30 °C and salinities of 5 and 30. Skeletonema costatum s.s. was identified as the species most adaptable to low-level salinity variations throughout its lifecycle.     

Colonization of Pythium oligandrum in the tomato rhizosphere for biological control of bacterial wilt disease analyzed by real-time PCR and confocal laser-scanning microscopy

It recently has been reported that the non-plant-pathogenic oomycete Pythium oligandrum suppresses bacterial wilt caused by Ralstonia solanacearum in tomato. As one approach to determine disease-suppressive mechanisms of action, we analyzed the colonization of P. oligandrum in rhizospheres of tomato using real-time polymerase chain reaction (PCR) and confocal laser-scanning microscopy. The real-time PCR specifically quantified P. oligandrum in the tomato rhizosphere that is reliable over a range of 0.1 pg to 1 ng of P. oligandrum DNA from 25 mg dry weight of soil. Rhizosphere populations of P. oligandrum from tomato grown for 3 weeks in both unsterilized and sterilized field soils similarly increased with the initial application of at least 5 x 10(5) oospores per plant. Confocal microscopic observation also showed that hyphal development was frequent on the root surface and some hyphae penetrated into root epidermis. However, rhizosphere population dynamics after transplanting into sterilized soil showed that the P. oligandrum population decreased with time after transplanting, particularly at the root tips, indicating that this biocontrol fungus is rhizosphere competent but does not actively spread along roots. Protection over the long term from root-infecting pathogens does not seem to involve direct competition. However, sparse rhizosphere colonization of P. oligandrum reduced the bacterial wilt as well as more extensive colonization, which did not reduce the rhizosphere population of R. solanacearum. These results suggest that competition for infection sites and nutrients in rhizosphere is not the primary biocontrol mechanism of bacterial wilt by P. oligandrum.     

Late arrival of Anguilla japonica glass eels at the Sagami River estuary in two recent consecutive year classes: ecology and socio-economic impacts

To determine the actual status of the recruiting glass eel stock of Anguilla japonica, we conducted a continuous monthly monitoring program for 2?years between November 2009 and October 2011 at the Sagami River estuary, Japan. A total of 114 and 372 A. japonica glass eels were observed in the 2009?C2010 and 2010?C2011 seasons, respectively. Recruitment patterns were the same in both years, starting in early winter (December in 2009 and November in 2010), increasing slightly until March, decreasing in April, but attaining the largest peak in June, after which no glass eels were observed during August?COctober. The Japanese eel has been known to spawn mainly in the summer, and glass eels recruit to their freshwater growth habitats during the winter to early spring. Our results clearly demonstrate an unexpected late arrival of glass eels in the early summer for two recent consecutive year classes. The summer recruitment found in our study indicates the unusual phenology of the Japanese eel, which may be a possible response to recent climate change.     

Comparison of the amount of thiotrophic symbionts in the deep-sea mussel <Emphasis Type="Italic">Bathymodiolus septemdierum</Emphasis> under different sulfide levels using fluorescent in situ hybridization

Various invertebrates inhabiting hydrothermal vents harbor thiotrophic endosymbionts that provide the host with nutrients and are possibly involved in the detoxification of harmful sulfides. In this study, we first determined the partial 16S rRNA gene sequence of the thiotrophic symbiont of the deep-sea mussel Bathymodiolus septemdierum, a dominant species at hydrothermal vents in the Izu–Ogasawara (Bonin) area. We then designed a new probe, Bsob692, for fluorescent in situ hybridization (FISH) using regions completely conserved among thiotrophic symbionts of all bathymodiolin mussels and established the protocol for FISH to compare the distribution and amount of the symbiont using an image analysis program that is commercially available. We compared fluorescent intensity in the gill of the mussels collected at different sites and found a higher intensity in specimens collected from a site with higher sulfide concentration. We also compared mussels reared in the presence and absence of sulfide and found that the former had a higher fluorescent intensity.     

Metabolite profiling of shoot extract,root extract,and root exudate of rice under nitrogen and phosphorus deficiency

ABSTRACT

Root exudate is derived from plant metabolites and its composition is affected by plant nutrient status. A deficiency of mineral nutrients, such as nitrogen (N) and phosphorus (P), strongly affects the type and amount of plant metabolites. We applied a metabolite profiling technique to investigate root exudates of rice plants under N and P deficiency. Oryza sativa was grown in culture solution containing two N levels (0 and 60 mg N L^?1) or two P levels (0 and 8 mg P L^?1). Shoot extracts, root extracts, and root exudates were obtained from the rice plants 5 and 15 days after transplanting and their metabolites were determined by capillary electrophoresis/time-of-flight mass spectrometry. Shoot N concentration and dry weight of rice plants grown at ?N level were lower than those of plants grown at +N level. Shoot P concentration and dry weight of rice plants grown at ?P level were lower than those of plants grown at +P level. One hundred and thirty-two, 127, and 98 metabolites were identified in shoot extracts, root extracts, and root exudates, respectively, at the two N levels. One hundred and thirty-two, 128, and 99 metabolites were identified in shoot extracts, root extracts, and root exudates, respectively, at the two P levels. Seventy-seven percent of the metabolites were exuded to the rhizosphere. The concentrations of betaine, gamma-aminobutyric acid, and glutarate in root exudates were higher at both ?N and ?P levels than at their respective high levels. The concentration of spermidine in root exudates was lower at both ?N and ?P levels than at their respective high levels. The concentrations of the other metabolites in root exudates were affected differently by plant N or P status. These results suggest that rice roots actively release many metabolites in response to N and P deficiency.