Takashi Yoshida

Isolation and characterization of a cyanophage infecting the toxic cyanobacterium Microcystis aeruginosa

ABSTRACT We isolated a cyanophage (Ma-LMM01) that specifically infects a toxic strain of the bloom-forming cyanobacterium Microcystis aeruginosa. Transmission electron microscopy showed that the virion is composed of anisometric head and a tail complex consisting of a central tube and a contractile sheath with helical symmetry. The morphological features and the host specificity suggest that Ma-LMM01 is a member of the cyanomyovirus group. Using semi-one-step growth experiments, the latent period and burst size were estimated to be 6 to 12 h and 50 to 120 infectious units per cell, respectively. The size of the phage genome was estimated to be ca. 160 kbp using pulse-field gel electrophoresis; the nucleic acid was sensitive to DNase I, Bal31, and all 14 restriction enzymes tested, suggesting that it is a linear double-stranded DNA having a low level of methylation. Phylogenetic analyses based on the deduced amino acid sequences of two open reading frames coding for ribonucleotide reductase alpha- and beta-subunits showed that Ma-LMM01 forms a sister group with marine and freshwater cyanobacteria and is apparently distinct from T4-like phages. Phylogenetic analysis of the deduced amino acid sequence of the putative sheath protein showed that Ma-LMM01 does not form a monophyletic group with either the T4-like phages or prophages, suggesting that Ma-LMM01 is distinct from other T4-like phages that have been described despite morphological similarity. The host-phage system which we studied is expected to contribute to our understanding of the ecology of Microcystis blooms and the genetics of cyanophages, and our results suggest the phages could be used to control toxic cyanobacterial blooms.

Ma-LMM01 Infecting Toxic Microcystis aeruginosa Illuminates Diverse Cyanophage Genome Strategies

Cyanobacteria and their phages are significant microbial components of the freshwater and marine environments. We identified a lytic phage, Ma-LMM01, infecting Microcystis aeruginosa, a cyanobacterium that forms toxic blooms on the surfaces of freshwater lakes. Here, we describe the first sequenced freshwater cyanomyovirus genome of Ma-LMM01. The linear, circularly permuted, and terminally redundant genome has 162,109 bp and contains 184 predicted protein-coding genes and two tRNA genes. The genome exhibits no colinearity with previously sequenced genomes of cyanomyoviruses or other Myoviridae. The majority of the predicted genes have no detectable homologues in the databases. These findings indicate that Ma-LMM01 is a member of a new lineage of the Myoviridae family. The genome lacks homologues for the photosynthetic genes that are prevalent in marine cyanophages. However, it has a homologue of nblA, which is essential for the degradation of the major cyanobacteria light-harvesting complex, the phycobilisomes. The genome codes for a site-specific recombinase and two prophage antirepressors, suggesting that it has the capacity to integrate into the host genome. Ma-LMM01 possesses six genes, including three coding for transposases, that are highly similar to homologues found in cyanobacteria, suggesting that recent gene transfers have occurred between Ma-LMM01 and its host. We propose that the Ma-LMM01 NblA homologue possibly reduces the absorption of excess light energy and confers benefits to the phage living in surface waters. This phage genome study suggests that light is central in the phage-cyanobacterium relationships where the viruses use diverse genetic strategies to control their hosts photosynthesis.

Ecological Dynamics of the Toxic Bloom-Forming Cyanobacterium Microcystis aeruginosa and Its Cyanophages in Freshwater

ABSTRACT The abundance of potentially Microcystis aeruginosa-infectious cyanophages in freshwater was studied using g91 real-time PCR. A clear increase in cyanophage abundance was observed when M. aeruginosa numbers declined, showing that these factors were significantly negatively correlated. Furthermore, our data suggested that cyanophage dynamics may also affect shifts in microcystin-producing and non-microcystin-producing populations.

Decomposition of nitric oxide on metallosilicates under a large excess oxygen condition with coexistence of a low concentration cetane

Catalytic removal of NO was conducted under the condition of large excess O2 by using different kinds of metallosilicates having MFI structure. With coexistence of a low concentration of cetane, H-Fe-silicate, which had both acidity and redox property, exhibited the maximum performance among the catalysts tested at a fairly lower temperature range, around 350 °C, and the NO was totally converted. The temperature dependence of NO conversion was consistently similar to that of cetane combustion.

Comptonized Photon Spectra of Supercritical Black Hole Accretion Flows with Application to Ultraluminous X-Ray Sources

Radiation spectra of supercritical black hole accretion flows are computed using a Monte Carlo method by post-processing the results of axisymmetric radiation hydrodynamic simulations. We take into account thermal/bulk Comptonization, free-free absorption, and photon trapping. We found that a shock-heated region (~108 K) appears at the funnel wall near the black hole where the supersonic inflow is reflected by the centrifugal barrier of the potential. Both thermal and bulk Comptonization significantly harden photon spectra although most of the photons upscattered above 40xa0keV are swallowed by the black hole due to the photon trapping. When the accretion rate onto the black hole is 200L E/c 2, where L E is the Eddington luminosity, the spectrum has a power-law component which extends up to ~10xa0keV by upscattering of photons in the shock-heated region. In higher mass accretion rates, the spectra roll over around 5xa0keV due to downscattering of the photons by cool electrons in the dense outflow surrounding the jet. Our results are consistent with the spectral features of ultraluminous X-ray sources, which typically show either a hard power-law component extending up to 10xa0keV or a rollover around 5xa0keV. We found that the spectrum of NGCxa01313 X-2 is quite similar to the spectrum numerically obtained for high accretion rate () source observed with low viewing angle (i = 10°-20°). Our numerical results also demonstrate that the face-on luminosity of supercritically accreting stellar mass black holes (10 M ☉) can significantly exceed 1040 erg s–1.

Diversity of viruses of the hyperthermophilic archaeal genus Aeropyrum, and isolation of the Aeropyrum pernix bacilliform virus 1, APBV1, the first representative of the family Clavaviridae ☆

We have surveyed the morphological diversity of viruses infecting the archaeon Aeropyrum pernix, the most thermophilic species among aerobic organisms, growing optimally at 90 degrees C, and isolated and characterized a novel virus, Aeropyrum pernix bacilliform virus 1, APBV1. This is the first virus to be described of the genus Aeropyrum and the archaeal order Desulfurococcales. The virion of APBV1 has rigid bacilliform morphology, about 140x20nm, with one end pointed and the other rounded. It contains highly glycosylated single major protein and three minor proteins. The circular, double-stranded DNA genome comprising 5278bp is the smallest for known archaeal viruses. None of the 14 putative genes, all on the same DNA strand, shows significant similarity to sequences in the public databases. The APBV1 infection caused neither retardation of host growth nor lysis of host cells, and integration of the viral genome into the host chromosome was not detected. On the basis of unusual morphological and genomic properties, we propose to consider APBV1 as the first representative of a new viral family, the Clavaviridae.

PURIFICATION AND CHARACTERIZATION OF A SULFOTRANSFERASE SPECIFIC TO N-21 OF SAXITOXIN AND GONYAUTOXIN 2+3 FROM THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM (DINOPHYCEAE)

A sulfotransferase (ST) specific to N‐21 of saxitoxin (STX) and gonyautoxin 2+3 (GTX2+3) designated as N‐ST was purified to homogeneity from the cytosolic fraction of clonal‐axenic vegetative cells of the toxic dinoflagellate Gymnodinium catenatum Graham GC21V, which causes paralytic shellfish poisoning. The enzyme transferred a sulfate group from 3′‐phosphoadenosine 5′‐phosphosulfate (PAPS) to N‐21 in the carbamoyl group of STX and GTX2+3 to produce GTX5 and C1+2, respectively. The molecular mass of the purified enzyme was determined by SDS‐PAGE to be 59 kDa. Gel filtration chromatography showed a native molecular mass of 65 kDa, indicating that the N‐ST is a monomeric enzyme. The N‐ST was specific to only N‐21 of STX and GTX2+3, and O‐22 sulfation was not observed. Moreover, the N‐ST was not active toward neo STX and GTX1+4, which differed from STX and GTX2+3, respectively, in only N‐1 hydroxylation. When various compounds previously reported to be substrates for STs in other organisms and paralytic shellfish poisoning toxins other than STX and GTX2+3 were added to the reaction mixture, N‐ST activity was not decreased. The enzyme required PAPS as the sole source of sulfate. The enzyme was optimally active at pH 6.0 and 25° C, and its activity was enhanced by Mg2+ and Co2+. The Km values of the N‐ST for STX and GTX2+3 were 16.1 μM and 29.8 μM, respectively.

Ardenticatena maritima gen. nov., sp. nov., a ferric iron- and nitrate-reducing bacterium of the phylum 'Chloroflexi' isolated from an iron-rich coastal hydrothermal field, and description of Ardenticatenia classis nov

A novel thermophilic, chemoheterotrophic, Gram-negative-staining, multicellular filamentous bacterium, designated strain 110S(T), was isolated from an iron-rich coastal hydrothermal field in Japan. The isolate is facultatively aerobic and chemoheterotrophic. Phylogenetic analysis using 16S rRNA gene sequences nested strain 110S(T) in a novel class-level clone cluster of the phylum Chloroflexi. The isolate grows by dissimilatory iron- and nitrate-reduction under anaerobic conditions, which is the first report of these abilities in the phylum Chloroflexi. The organism is capable of growth with oxygen, ferric iron and nitrate as a possible electron acceptor, has a wide range of growth temperatures, and tolerates higher NaCl concentrations for growth compared to the other isolates in the phylum. Using phenotypic and phylogenetic data, strain 110S(T) (= JCM 17282(T) = NBRC 107679(T) = DSM 23922(T) = KCTC 23289(T) = ATCC BAA-2145(T)) is proposed as the type strain of a novel species in a new genus, Ardenticatena maritima gen. nov., sp. nov. In addition, as strain 110S(T) apparently constitutes a new class of the phylum Chloroflexi with other related uncultivated clone sequences, we propose Ardenticatenia classis nov. and the subordinate taxa Ardenticatenales ord. nov. and Ardenticatenaceae fam. nov.

Defective control and adaptation of reflex eye movements in mutant mice deficient in either the glutamate receptor δ2 subunit or Purkinje cells

The ionotropic glutamate receptor δ2 subunit (GluRδ2) is selectively expressed in cerebellar Purkinje cells and is implicated in long‐term depression, synaptic formation and elimination. To study the effect of GluRδ2 deficiency on motor control, we measured the vestibulo‐ocular reflex (VOR) and optokinetic response (OKR) induced by sinusoidal rotation of the animal and/or the surrounding screen in two GluRδ2 mutant mice: a GluRδ2 knockout mouse (δ2–/–) and a lurcher mouse with a point mutation in the GluRδ2 gene resulting in loss of all Purkinje cells. δ2–/– showed significantly higher VOR gain in the dark (VORD) than in the wild‐type. In δ2–/–, the VOR gain in light was lower than that in the dark. The phase of OKR lagged more in δ2–/– than in lurcher and wild‐type mice. Both mutant mice failed to change the VORD or OKR gain adaptively in response to sustained vestibular and/or visual stimulation. Basal properties of VOR and OKR changed little by lesion of the flocculus, but they changed substantially by lesion of the inferior olivary nuclei (IO). The abnormal VOR gain and OKR phase delay were clearly reduced in δ2–/– by the latter lesion. Our results indicate that failures in the GluRδ2‐dependent synaptic regulation affect motor performance more severely than loss of cerebellar cortical outputs. This study suggests that the anomalies in δ2–/– are dependent on inputs from IO and that GluRδ2 deficiency changed properties of not only the cerebellar cortex but also the brainstem neuronal pathways controlling reflex eye movements during development.

No Decomposition on Cu-Incorporated A-Zeolites under the Reaction Condition of Excess Oxygen with a Small Amount of Hydrocarbons

Summary Copper containing A-type zeolites which contained copper with considerably high concentrations were synthesized through crystallization. It was confirmed that Cu + ions in the crystals could be stably maintained compared with those in the copper-loaded samples prepared by an ion-exchanged method. NO decomposition activity on the Cu-A catalyst corresponded to the capacity of redox response. Even under an excess oxygen condition the NO decomposition progressed smoothly at around 300 – 350° by the addition of a very small concentration of n-C 8 - n-C 16 saturated hydrocarbons. Especially cetane (n-C 16 ) exhibited the marked effect, and NO was decomposed completely at that temperature range. To explain these unusual non-linear reaction phenomena, Microscopic Sequential Reaction mechanism was proposed and the necessary conditions to realize this mechanism were discussed.