Purification and Properties of an Amylase from Bacillus cereus NY-14

An extracellular amylase was obtained from a culture medium of Bacillus cereus NY-14. This enzyme was purified to show a single band on disc gel electrophoresis by ammonium sulfate fractionation and Sephadex G-100 gel filtration to 1101-fold of the activity of the original culture liquor. The purified enzyme had a molecular weight of 55,000, an isoelectric point of 6.13, an optimum pH of 6.0, and an optimum temperature of 55°C. The pH-stability range was wide; the enzyme retained more than 80% of its initial activity in the range of pH 5.5 to 12. It was stable below 35°C and required calcium ions for the stabilization. The action pattern of this enzyme on amylaceous polysaccharides was unique in that the predominant product was maltopentaose. The purified amylase could also digest starch granules of such plants as rice, barley, corn, and kuzu to produce maltopentaose as the main product.     

Volatilization of mercury under acidic conditions from mercury-polluted soil by a mercury-resistant Acidithiobacillus ferrooxidans SUG 2-2.

Volatilization of mercury under acidic conditions from soil polluted with mercuric chloride (1.5 mg Hg/kg soil) was studied with resting cells of a mercury-resistant strain, Acidithiobacillus ferrooxidans SUG 2-2. When resting cells of SUG 2-2 (0.01 mg of protein) were incubated for 10 d at 30 degrees C in 20 ml of 1.6 mM sulfuric acid (pH 2.5) with ferrous sulfate (3%) and mercury-polluted soil (1 g), which contained 7.5 nmol of Hg, approximately 4.1 nmol of mercury was volatilized, indicating that 54% of the total mercury in the soil was volatilized. The amount of mercury volatilized from the soil was dependent on the concentration of Fe2+ added to the medium. When elemental sulfur, sodium tetrathionate, and pyrite were used as an electron donor for the mercury reduction, 16, 2.4 and 0.84%, respectively, of the total mercury added to the solution were volatilized. The optimum pH and temperature for mercury volatilization were 2.5 and 30 degrees C. Approximately 92% of the total mercury in a salt solution (pH 2.5) with resting cells of SUG 2-2 (0.01 mg of protein), ferrous sulfate (3%) and mercury-polluted soil (1 g) was volatilized by further addition of both resting cells and Fe2+ and by incubating for 30 d at 30 degrees C.     

Feeding strategies of Japanese monkeys against deterioration of habitat quality

Field observations of the feeding behaviour of Japanese monkeys were carried out from autumn to winter on Kinkazan Island which is covered with cool temperate forest. As a result, the following two points became clear: (1) the available food items were fixed for a long time; and (2) the habitat quality deteriorated monotonously because the monkeys themselves or their competitors, such as wild mice, utilized the food resources. Against the decrease in food intake caused by this deterioration of the habitat quality, the monkeys controlled the decrease in food intake by employing the following strategies: (1) they recovered their feeding speed by exploiting new food patches (patch-increase strategy); (2) they extended the time spent on feeding (time-extension strategy); and (3) they changed their food (food-change strategy). The former two strategies operated earlier than the third one.     

Fungal ecdysteroid-22-oxidase, a new tool for manipulating ecdysteroid signaling and insect development

Steroid hormones ecdysteroids regulate varieties of developmental processes in insects. Although the ecdysteroid titer can be increased experimentally with ease, its artificial reduction, although desirable, is very difficult to achieve. Here we characterized the ecdysteroid-inactivating enzyme ecdysteroid-22-oxidase (E22O) from the entomopathogenic fungus Nomuraea rileyi and used it to develop methods for reducing ecdysteroid titer and thereby controlling insect development. K(m) and K(cat) values of the purified E22O for oxidizing ecdysone were 4.4 μM and 8.4/s, respectively, indicating that E22O can inactivate ecdysone more efficiently than other ecdysteroid inactivating enzymes characterized so far. The cloned E22O cDNA encoded a FAD-dependent oxidoreductase. Injection of recombinant E22O into the silkworm Bombyx mori interfered with larval molting and metamorphosis. In the hemolymph of E22O-injected pupae, the titer of hormonally active 20-hydroxyecdysone decreased and concomitantly large amounts of inactive 22-dehydroecdysteroids accumulated. E22O injection also prevented molting of various other insects. In the larvae of the crambid moth Haritalodes basipunctalis, E22O injection induced a diapause-like developmental arrest, which, as in normal diapause, was broken by chilling. Transient expression of the E22O gene by in vivo lipofection effectively decreased the 20-hydroxyecdysone titer and blocked molting in B. mori. Transgenic expression of E22O in Drosophila melanogaster caused embryonic morphological defects, phenotypes of which were very similar to those of the ecdysteroid synthesis deficient mutants. Thus, as the first available simple but versatile tool for reducing the internal ecdysteroid titer, E22O could find use in controlling a broad range of ecdysteroid-associated developmental and physiological phenomena.     

Genus-specific and phase-dependent effects of nitrate on a sulfate-reducing bacterial community as revealed by dsrB-based DGGE analyses of wastewater reactors

The biogenic production of hydrogen sulfide is a serious problem associated with wastewater treatment. The aim of this study was to investigate the inhibitory effect of nitrate on the dynamics of sulfate-reducing bacteria (SRB) community in a laboratory-scale wastewater reactor, originating from a denitrifying plant using activated sludge. For this purpose, denaturing gradient gel electrophoresis (DGGE) analysis targeting the dsrB (dissimilatory sulfite reductase) gene was used in combination with chemical analyses and measurement of oxidation and reduction potential (ORP). The reactors were initially dosed with 1.0 and 4.0 g/L potassium nitrate and anaerobically incubated for 490 h. Addition of 4.0 g/L nitrate to the reactor was associated with a prolonged inhibition (over 300 h, i.e., 12.5 days) of sulfate reduction and this was consistent with a rapid decrease in ORP associated with nitrate depletion. The DGGE analysis revealed that nitrate addition remarkably attenuated a distinct group of dsrB related to Desulfovibrio, whereas other dsrB groups were not influenced. Furthermore, another sulfate reduction by Syntrophobacter in the later stages of the incubation period occurred in both reactors (regardless of the nitrate concentration), suggesting that different SRB groups are associated with sulfate reduction at different stages of the wastewater treatment process.     

The AddAB helicase/nuclease forms a stable complex with its cognate chi sequence during translocation

The Bacillus subtilis AddAB enzyme possesses ATP-dependent helicase and nuclease activities, which result in the unwinding and degradation of double-stranded DNA (dsDNA) upon translocation. Similar to its functional counterpart, the Escherichia coli RecBCD enzyme, it also recognizes and responds to a specific DNA sequence, referred to as Chi (chi). Recognition of chi triggers attenuation of the 3'- to 5'-nuclease, which permits the generation of recombinogenic 3'-overhanging, single-stranded DNA (ssDNA), terminating at chi. Although the RecBCD enzyme briefly pauses at chi, no specific binding of RecBCD to chi during translocation has been documented. Here, we show that the AddAB enzyme transiently binds to its cognate chi sequence (chi(Bs): 5'-AGCGG-3') during translocation. The binding of AddAB enzyme to the 3'-end of the chi(Bs)-specific ssDNA results in protection from degradation by exonuclease I. This protection is gradually reduced with time and lost upon phenol extraction, showing that the binding is non-covalent. Addition of AddAB enzyme to processed, chi(Bs)-specific ssDNA that had been stripped of all protein does not restore nuclease protection, indicating that AddAB enzyme binds to chi(Bs) with high affinity only during translocation. Finally, protection of chi(Bs)-specific ssDNA is still observed when translocation occurs in the presence of competitor chi(Bs)-carrying ssDNA, showing that binding occurs in cis. We suggest that this transient binding of AddAB to chi(Bs) is an integral part of the AddAB-chi(Bs) interaction and propose that this molecular event underlies a general mechanism for regulating the biochemical activities and biological functions of RecBCD-like enzymes.     

GINS is a DNA polymerase epsilon accessory factor during chromosomal DNA replication in budding yeast

GINS is a protein complex found in eukaryotic cells that is composed of Sld5p, Psf1p, Psf2p, and Psf3p. GINS polypeptides are highly conserved in eukaryotes, and the GINS complex is required for chromosomal DNA replication in yeasts and Xenopus egg. This study reports purification and biochemical characterization of GINS from Saccharomyces cerevisiae. The results presented here demonstrate that GINS forms a 1:1 complex with DNA polymerase epsilon (Pol epsilon) holoenzyme and greatly stimulates its catalytic activity in vitro. In the presence of GINS, Pol epsilon is more processive and dissociates more readily from replicated DNA, while under identical conditions, proliferating cell nuclear antigen slightly stimulates Pol epsilon in vitro. These results strongly suggest that GINS is a Pol epsilon accessory protein during chromosomal DNA replication in budding yeast. Based on these results, we propose a model for molecular dynamics at eukaryotic chromosomal replication fork.