Synthesis of Short-chain-length/Medium-chain-length Polyhydroxyalkanoate (PHA) Copolymers in Peroxisome of the Transgenic Arabidopsis Thaliana Harboring the PHA Synthase Gene from Pseudomonas sp. 61-3

In this paper, the photosynthetic production of short-chain-length/medium-chain-length polyhydroxyalkanoate (PHA) copolymers is reported. The wild-type and highly active doubly mutated PHA synthase 1 (S325T/Q481K, abbreviated ST/QK) genes from Pseudomonas sp. 61-3 were introduced into Arabidopsis thaliana. Peroxisome targeting signal 1 (PTS1) was used to target PHA synthases into the peroxisome to synthesize PHA from the intermediates of the β-oxidation pathway. The transgenic Arabidopsis produced PHA copolymers consisting of 40–57 mol% 3-hydroxybutyrate, 21–49 mol% 3-hydroxyvalerate, 8–18 mol% 3-hydroxyhexanoate, and 2–8 mol% 3-hydroxyoctanoate. The maximum PHA contents were 220μ g/g cell dry weight (cdw) in leaves, and 36μ g/g cdw in stems, respectively. The expression of the ST/QK mutated PHA synthase in leaves gene did not lead to significant difference in PHA content and monomer composition of PHAs, compared to the wild-type PHA synthase gene, suggesting that the supply of monomers may be a rate-determining step of PHA biosynthesis in the peroxisome. However, in stems, there were significant differences dependent on whether the wild-type or ST/QK mutated PHA synthase was expressed. These results suggest that tissue-specific monomer availability is important in determining the final mol% composition of PHA copolymers produced by the peroxisome in plants.     

Spiroplasma infection causes either early or late male killing in <Emphasis Type="Italic">Drosophila</Emphasis>, depending on maternal host age

Symbiont-induced male-killing phenotypes have been found in a variety of insects. Conventionally, these phenotypes have been divided into two categories according to the timing of action: early male killing at embryonic stages and late male killing at late larval stages. In Drosophila species, endosymbiotic bacteria of the genus Spiroplasma have been known to cause early male killing. Here, we report that a spiroplasma strain normally causing early male killing also induces late male killing depending on the maternal host age: male-specific mortality of larvae and pupae was more frequently observed in the offspring of young females. As the lowest spiroplasma density and occasional male production were also associated with newly emerged females, we proposed the density-dependent hypothesis for the expression of early and late male-killing phenotypes. Our finding suggested that (1) early and late male-killing phenotypes can be caused by the same symbiont and probably by the same mechanism; (2) late male killing may occur as an attenuated expression of early male killing; (3) expression of early and late male-killing phenotypes may be dependent on the symbiont density, and thus, could potentially be affected by the host immunity and regulation; and (4) early male killing and late male killing could be alternative strategies adopted by microbial reproductive manipulators.     

Application of a Larval Medaka Assay to Evaluate the Fish Safety Level in Sagami River, Japan

Physico-chemical characteristics of some river and hand-dug well waters used for drinking and domestic purposes in the oil rich Niger Delta area of Nigeria were assessed using standard methods. The concentrations of the parameters in the river water samples ranged in the following order: pH (5.6–6.9), temperature (26.90–28.60°C), turbidity (23–63 NTU), electrical conductivity (52–184 μs/cm), DO (5.4–7.2 mg/l), BOD (21–57 mg/l), TDS (6.0–217 mg/l), PO₄ ³⁻ (0.19–1.72 mg/l), SO₄ ²⁻ (25–36.8 mg/l), NO₃ ⁻ (20.3–28 mg/l), Fe (6.07–15.71 mg/l), Zn (0.04–0.24 mg/l), Pb (0.01–0.17 mg/l), Ni (0.01–0.13 mg/l), Vn (0.01–0.20 mg/l) and Hg (0.001–0.002 mg/l). The concentrations of these parameters in the hand-dug well water ranged in the following order: pH (5.7–6.8) temperature (26–30°C), turbidity (134–171 NTU), electrical conductivity (160–340 μs/cm), DO (5.4–6.4 mg/l), BOD (13–34 mg/l), TDS (110–190 mg/l), PO₄ ³⁻ (0.84–1.84 mg/l), SO₄ ²⁻ (10.6–28.1 mg/l), NO₃ ⁻ (11.3–23 mg/l), Fe (13.17–16.31 mg/l), Ni (0.01–0.02 mg/l), Vn (0.01–0.04 mg/l) and Hg (0.001–0.004 mg/l). The concentrations of BOD, turbidity, NO₃ ⁻ and Fe in the water samples were above WHO and FMENV permissible limits for safe drinking water. The results suggest that the use of such waters for drinking and domestic purposes pose a serious threat to the health of the users and calls for the intervention of government agencies.     

Evaluating the fish safety level of river water and wastewater with a larval medaka assay

A larval medaka (Oryzias latipes) assay was proposed to evaluate the fish safety level of river waters and wastewaters. Organic toxicants were 10-100 times concentrated with adsorption cartridges from 4 l of river water or 1-10 times concentrated from 400 ml of wastewater. Toxicity of these concentrated solutions was determined by exposing 48-72 h post-hatch age larvae for 48 h. The method effectively revealed a variation of the median lethal concentration ratio (LCR50) from 13 to >100 in 125 river water samples, and from <1 to >10 in five typical wastewater samples. Ayase River, which takes water mostly from agricultural or household discharge, showed significantly (P<0.001) lower LCR50 than Sagami River that takes natural water as the source. Safety Levels in both Sagami River and Ayase River were influenced by the irrigation activity, LCR50 at some sites showing a seasonal-specific decrease in winter. Pollution from pulp and paper industries contributed to the low LCR50 in several tributaries of Ayase River. Required little manpower in sampling, pretreatment and testing, the proposed larval medaka assay was proved as an efficient tool for screening those high risk sites for priority management.     

Degradation activity of Clostridium species DC-1 in the cis-1,2-dichloroethylene contaminated site in the presence of indigenous microorganisms and Escherichia coli

We showed the cis-1,2-dichloroethylene (cis-1,2-DCE) dechlorination ability of Clostridium species DC-1 in association with other bacteria. Result of denaturing gradient gel electrophoresis showed the dominant band pattern of DC-1 during the degradation time of cis-1,2-DCE and dominance of some other Clostridium species. Experiment with addition of Escherichia coli (E. coli) showed that dechlorinating activity of DC-1 was not inhibited by the presence of E. coli. Pour plate experiment with DC-1 and E. coli revealed that the dominance of Clostridium species caused the decrease of E. coli growth in a bioremediation state. This result suggested the possibility of Clostridium species DC-1 as a degrader of cis-1,2-DCE, in a cis-1,2-DCE contaminated site where an indigenous microbial community is present. Experiment conducting with E. coli suggested that the strain in the contaminated site did not inhibit the degradation of cis-1,2-DCE and during the degradation period, rather some other Clostridium species became dominant and the growth of E. coli would be decreased. This finding could be a very positive approach for implementing the dechlorinating bacteria at aliphatic chlorinated component contaminated sites.     

Purification and characterization of extracellular poly(3-hydroxybutyrate) depolymerases

Five extracellular PHB depolymerases of bacteria isolated from various sources were purified to electrophoretic homogeneity and compared with known extracellular PHB depolymerase fromAlcaligenes faecalis T1. The molecular mass of these enzymes were all around 40–50 kDa. Nonionic detergent, diisopropylfluorophosphate and dithiothreitol inhibited the PHB depolymerase activity of all these enzymes. Trypsin abolished PHB depolymerase activity, but not theD-3-hydroxybutyric acid dimer hydrolase activity of all the enzymes. These results showed that the basic properties of these PHB depolymerases resemble those of theA. faecalis T1 enzyme. Analysis ofN-terminal amino acid sequence of the purified enzymes revealed that these enzymes includingA. faecalis T1 enzyme fall into three groups.     

Ultrastructural alterations on chloride cells of the yellowtail Seriola quinqueradiata,following exposure to the red tide species Chattonella antiqua

By means of scanning and transmission electron microscopy, the influence of the red tide species Chattonella antiqua was examined with respect to the surface ultrastructures of chloride cells of the yellowtail Seriola quinqueradiata. Conspicuous ultrastructural alterations occurred on the apical surface of these cells. The majority of chloride cells in the control gills showed an apical surface with numerous cellular extensions, while more than half the chloride cells affected by red tide organisms exhibited an apical surface with fewer and smaller extensions, a wrinkled apical surface, or a protruded apical surface. These ultrastructural alterations of chloride cell surface may be due to the partial disturbance of salinity by C. antiqua, and reflect the changes of the ion-transport function in yellowtail gills exposed to red tide water.