Effective isolation of bacterioplankton genus Polynucleobacter from freshwater environments grown on photochemically degraded dissolved organic matter

Effective isolation of freshwater bacterioplankton belonging to genus Polynucleobacter from a shallow eutrophic lake and its tributary was achieved by size-selective filtration with a 0.7-μm pore filter and cultivation on R2A agar medium. Partial 16S rRNA gene analysis showed that over 80% of all the strains were highly similar to the Polynucleobacter cluster. Essential medium components for effective cultivation are pyruvate, yeast extract and peptone, whereas soluble starch and glucose are not necessary. Isolate KF001 (affiliated with Polynucleobacter subcluster D) has a strict requirement for organic acids as carbon sources, and we hypothesize that the Polynucleobacter cluster of bacteria could utilize compounds formed via photochemically dissolved organic matter (DOM) degradation for growth. Because organic acids form from solar radiation of DOM in aquatic environments, carbon sources that are typical products of DOM photochemical degradation were added to the medium. These compounds were readily utilized by KF001 in this study. Finally, we observed the stimulation of strain KF001 activity by photochemical degradation of natural lake water. Our findings suggest a carbon flow of DOM photoproducts to Polynucleobacter in the freshwater microbial loop.     

Cross-reactivity in rapid diagnostic tests between human malaria and zoonotic simian malaria parasite Plasmodium knowlesi infections

Plasmodium knowlesi has a relatively broad host range extending to humans, in whom it causes zoonotic malaria. Recent studies have shown that human infection with P. knowlesi is widely distributed in forested areas of Southeast Asia. In the present study, we evaluated commercial rapid diagnostic tests (RDTs) for human malaria to assess their reactivity and sensitivity in detecting P. knowlesi parasites using blood samples obtained from infected monkeys. The blood samples were assayed using two commercial RDTs based on immunochromatographic assays: (i) the OptiMAL-IT, designed to detect parasite lactate dehydrogenase (pLDH) of both P. falciparum and other plasmodia, and (ii) the Entebe Malaria Cassette (MC), designed to detect P. falciparum-specific histidine-rich protein 2 (PfHRP2) and P. vivax-specific pLDH. Interestingly, when the P. knowlesi-infected blood samples were examined with the RDTs, OptiMAL test results were interpreted as falciparum malaria-positive, while Entebe MC test results were interpreted as vivax malaria-positive. The sensitivities of both tests in detecting P. knowlesi parasite were similar to those for P. falciparum and higher than P. vivax. Thus, commercial RDTs based on detection of pLDH should be used with great caution, and should not replace conventional microscopy in the diagnosis of suspected cases of P. knowlesi malaria.     

Potential impacts of the invasive flatworm Platydemus manokwari on arboreal snails

The introduction of the snail-eating flatworm Platydemus manokwari (Tricladida: Rhynchodemidae) has been considered a cause of the extinction of native land snails on several Pacific islands. Although P. manokwari is known to attack land snails on the ground, whether P. manokwari attacks snails on trees remains unclear. To clarify the effect of P. manokwari on arboreal snails, we examined survival rates of land snails experimentally placed on tree trunks (0.5–2.0 m above the ground) in a forest on Chichijima, Ogasawara (Bonin) Islands, in the northwestern Pacific Ocean. The survival of snails experimentally placed on tree trunks with artificially created snail scent trails rapidly decreased for 7 days, and the mortality was caused by P. manokwari predation. However, snails placed on tree trunks without snail scent trails were not attacked by P. manokwari. Therefore, P. manokwari climbed tree trunks, likely tracking the snail scent. We found that over 40% of the snails placed on tree trunks with snail scent trails were eaten by P. manokwari within 7 days. This experiment supports the hypothesis that P. manokwari predation is an important cause of the rapid decline or extinction of native arboreal snails on Pacific islands.     

A new high-alkaline alginate lyase from a deep-sea bacterium Agarivorans sp.

A high-alkaline, salt-activated alginate lyase is produced by Agarivorans sp. JAM-A1m from a deep-sea sediment off Cape Nomamisaki on Kyushu Island, Japan. Purified to homogeneity, as judged by SDS-PAGE, the enzyme (A1m) had a molecular mass of approximately 31 kDa. The optimal pH was around 10 in glycine–NaOH buffer, and the activity was increased to 1.8 times by adding 0.2 M NaCl. However, when the optimal pH in the presence of 0.2 M NaCl was shifted to pH 9.0, the activity was more than 10 times compared with that at pH 9 in the absence of NaCl. A1m showed the optimal temperature at around 30°C and was stable to incubation between pH 6 and 9. The enzyme degraded favorably mannuronate–guluronate and guluronate-rich fragments in alginate. Shotgun cloning and sequencing of the gene for A1m revealed a 930-bp open reading frame, which encoded a mature enzyme of 289 amino acids (32,295 Da) belonging to polysaccharide lyase family 7. The deduced amino acid sequence showed the highest similarity to that of a Klebsiella enzyme, with only 54% identity.     

Complex coevolutionary history of symbiotic Bacteroidales bacteria of various protists in the gut of termites

Background  

The microbial community in the gut of termites is responsible for the efficient decomposition of recalcitrant lignocellulose. Prominent features of this community are its complexity and the associations of prokaryotes with the cells of cellulolytic flagellated protists. Bacteria in the order Bacteroidales are involved in associations with a wide variety of gut protist species as either intracellular endosymbionts or surface-attached ectosymbionts. In particular, ectosymbionts exhibit distinct morphological patterns of the associations. Therefore, these Bacteroidales symbionts provide an opportunity to investigate not only the coevolutionary relationships with the host protists and their morphological evolution but also how symbiotic associations between prokaryotes and eukaryotes occur and evolve within a complex symbiotic community.     

Characterization of discontinuous epitope of prion protein recognized by the monoclonal antibody T2

The anti-prion protein (PrP) monoclonal antibody T2 has previously been prepared using PrP-knockout mice immunized with mouse recombinant PrP residues 121-231, however its interaction mechanism to PrP antigen has not been cleared. Here we identified and characterized the epitope of T2 antibody. The competitive ELISA with 20-mer synthetic peptides derived from PrP121-231 showed that T2 antibody had no affinity for these peptides. The analysis with deletion mutants of PrP revealed that 10 amino acids in the N terminus and 66 amino acids in the C terminus of PrP121-231 were necessary for reactivity with T2. Two far regions are necessary for complete affinity of the T2 antibody for PrP; either region alone is not sufficient to retain the affinity. The epitope recognized by T2 antibody is discontinuous and conformational. We examined the effect of disulfide bond and salt bridges. Alkylation of cysteine residues in C terminus of PrP121-231, which breaks a disulfide bond and disrupts the structure, had diminished the reactivity. Mutations induced in the PrP121-231 to break the disulfide bond or salt bridges, markedly had reduced the reactivity with T2 antibody. It suggests that T2 antibody recognized the structure maintained by the disulfide bond and salt bridges.     

Intraspecies prion transmission results in selection of sheep scrapie strains

Background

Sheep scrapie is caused by multiple prion strains, which have been classified on the basis of their biological characteristics in inbred mice. The heterogeneity of natural scrapie prions in individual sheep and in sheep flocks has not been clearly defined.

Methodology/Principal Findings

In this study, we intravenously injected 2 sheep (Suffolk and Corriedale) with material from a natural case of sheep scrapie (Suffolk breed). These 3 sheep had identical prion protein (PrP) genotypes. The protease-resistant core of PrP (PrPres) in the experimental Suffolk sheep was similar to that in the original Suffolk sheep. In contrast, PrPres in the Corriedale sheep differed from the original PrPres but resembled the unusual scrapie isolate, CH1641. This unusual PrPres was not detected in the original sheep. The PrPres distributions in the brain and peripheral tissues differed between the 2 breeds of challenged sheep. A transmission study in wild-type and TgBoPrP mice, which overexpressing bovine PrP, led to the selection of different prion strains. The pathological features of prion diseases are thought to depend on the dominantly propagated strain.

Conclusions/Significance

Our results indicate that prion strain selection occurs after both inter- and intraspecies transmission. The unusual scrapie prion was a hidden or an unexpressed component in typical sheep scrapie.     

Human Artificial Chromosome with a Conditional Centromere for Gene Delivery and Gene Expression

Human artificial chromosomes (HACs), which carry a fully functional centromere and are maintained as a single-copy episome, are not associated with random mutagenesis and offer greater control over expression of ectopic genes on the HAC. Recently, we generated a HAC with a conditional centromere, which includes the tetracycline operator (tet-O) sequence embedded in the alphoid DNA array. This conditional centromere can be inactivated, loss of the alphoid^tet-O (tet-O HAC) by expression of tet-repressor fusion proteins. In this report, we describe adaptation of the tet-O HAC vector for gene delivery and gene expression in human cells. A loxP cassette was inserted into the tet-O HAC by homologous recombination in chicken DT40 cells following a microcell-mediated chromosome transfer (MMCT). The tet-O HAC with the loxP cassette was then transferred into Chinese hamster ovary cells, and EGFP transgene was efficiently and accurately incorporated into the tet-O HAC vector. The EGFP transgene was stably expressed in human cells after transfer via MMCT. Because the transgenes inserted on the tet-O HAC can be eliminated from cells by HAC loss due to centromere inactivation, this HAC vector system provides important novel features and has potential applications for gene expression studies and gene therapy.     

Protein expression during Flammulina velutipes fruiting body formation

Formation of the Flammulina velutipes fruiting body can be induced by lowering the ambient temperature (first treatment) in complete darkness. Fruiting bodies formed under these conditions elongate without pileus formation (pinhead fruiting body), suggesting that they cannot mature in complete darkness. However, after light treatment of the pinhead fruiting body (second treatment), a pileus develops immediately, and the stipe also thickens and becomes increasingly pigmented. The apical region swells as a result of cell division starting 2 days after light treatment, the pileus–stipe junction fracture and hymenium primordia form on day 4, and gills appear at day 6. Pf1 and Pf3 are specifically expressed after exposure to low temperature without light. The cell wall-associated protein [pileus-specific hydrophobin-like protein (PSH)] is specifically induced in the pileus, but not in the stipe, following the second light treatment to the pinhead fruiting body. These results suggest that Pf1 and Pf3 would be involved in fruiting body induction and that PSH would be involved in pileus formation. These phenomena will aid further histological and molecular biological investigations into the mechanisms behind fruiting body development in F. velutipes.