Cloning and expression of a gene encoding a novel thermostable thiocyanate-degrading enzyme from a mesophilic alphaproteobacteria strain THI201.

Strain THI201, a member of the alphaproteobacteria, is a novel thiocyanate (SCN(-))-degrading bacterium isolated from lake water enriched with potassium thiocyanate (KSCN). This bacterium carries the enzyme thiocyanate hydrolase (SCNase) that hydrolyses thiocyanate to carbonyl sulfide and ammonia. Characterization of both native and recombinant SCNase revealed properties different from known SCNases regarding subunit structure and thermostability: SCNase of strain THI201 was composed of a single protein and thermostable. We cloned and sequenced the corresponding gene and determined a protein of 457 amino acids of molecular mass 50 267 Da. Presence of a twin-arginine (Tat) signal sequence of 32 amino acids was found upstream of SCNase. The deduced amino acid sequence of SCNase showed 83% identity to that of a putative uncharacterized protein of Thiobacillus denitrificans ATCC 25259, but no significant identity to those of three subunits of SCNase from Thiobacillus thioparus strain THI115. The specific activities of native and recombinant enzyme were 0.32 and 4-15 µmol min(-1) (mg protein)(-1), respectively. The maximum activity of SCNase was found in the temperature range 30-70 °C. The thiocyanate-hydrolysing activity in both enzymes was decreased by freeze-thawing, although 25-100% of the activity of recombinant protein could be retrieved by treating the enzyme at 60 °C for 15 min. Furthermore, both native and recombinant enzymes retained the activity after pre-treatment of the protein solution at temperatures up to 70 °C.

Serum-free culture of an embryonic cell line from Bombyx mori and reinforcement of susceptibility of a recombinant BmNPV by cooling.

We established the first continuous cell line that uses a serum-free culture from the embryo of Bombyx mori (Lepidoptera: Bombycidae), designated as NIAS-Bm-Ke17. This cell line was serially subcultured in the SH-Ke-117 medium. The cells adhere weakly to the culture flask, and most cells have an oval shape. The cell line was subcultured 154 times, and the population doubling time is 83.67±5.22 h. Random amplification of polymorphic DNA-polymerase chain reaction with a tenmar single primer for discrimination of insect cell lines recognized the NIAS-Bm-Ke1 cell line as B. mori. This cell line does not support the growth of the B. mori nuclear polyhedrosis virus (BmNPV) in the absence of the heat-inactivated hemolymph of B. mori. However, the heat-inactivated hemolymph in 1% volume of the medium supported a high level of susceptibility to BmNPV. In addition, the cooling treatment of the cells at 2.5°C also enhanced the susceptibility. We report a new serum-free culture system of the B. mori cell line for the baculovirus expression vector system.

Input-output analysis of in vivo photoassimilate translocation using Positron-Emitting Tracer Imaging System (PETIS) data.

The Positron-Emitting Tracer Imaging System (PETIS) is introduced for monitoring the distribution of (11)C-labelled photoassimilates in Sorghum. The obtained two-dimensional image data were quantitatively analysed using a transfer function analysis approach. While one half of a Sorghum root in a split root system was treated with either 0, 100, or 500 mM NaCl dissolved in the nutrient solution, tracer images of the root halves and the lower stem section were recorded using PETIS. From the observed tracer levels, parameters were estimated, from which the mean speed of tracer transport and the proportion of tracer moved between specified image positions were deduced. Transport speed varied between 0.7 and 1.8 cm min(-1) with the difference depending on which part of the stem was involved. When data were collected in the lowest 0.5-1 cm of the stem, which included the point where the roots emerge, transport speed was less. Rapid changes in NaCl concentration, from 0 to 100 mM, resulted in short-term increases of assimilate import into the treated root. This response represented a transient osmotic effect, that was compensated for in the medium-term by osmotic adaptation. Higher concentrations of NaCl (500 mM) resulted in distinctly less photoassimilate transport into the treated root half. The present results agree with earlier observations, showing that transport of (11)C-labelled photoassimilates measured with the PETIS detector system can be quantified using the method of input-output analysis. It is worth noting that with the PETIS detector system, areas of interest do not need to be defined until after data collection. This means that unexpected behaviour of a plant organ will be seen, which is not necessarily the case with conventional detector systems looking at predefined areas of interest.

Quick and reversible inhibition of soybean root nodule growth by nitrate involves a decrease in sucrose supply to nodules.

The upper part of a nodulated soybean root hydroponically cultured in a glass bottle was monitored using a computer microscope under controlled environmental conditions, and the diameter of individual nodules was measured from 10-24 d after planting. The diameter of a root nodule attached to the primary root increased from 1 mm to 6 mm for 2 weeks under nitrogen-free conditions. The increase in diameter of the nodules was almost completely stopped after 1 d of supplying 5 mM nitrate, and was due to the cessation of nodule cell expansion. However, nodule growth quickly returned to the normal growth rate following withdrawal of nitrate from the solution. The reversible depression of nodule growth by nitrate was similar to the restriction of photoassimilate supply by continuous dark treatment for 2 d followed by normal light/dark conditions. In addition, the inhibitory effect of nitrate was partially alleviated by the addition of 3% (w/v) sucrose to the medium. Plant leaves were exposed to (11)C or (14)C-labelled carbon dioxide to investigate the effects of 5 mM nitrate on the translocation and distribution of photosynthates to nodules and roots. Supplying 5 mM nitrate stimulated the translocation rate and the distribution of labelled C in nitrate-fed parts of the roots. However, the (14)C partitioning to nodules decreased from 9% to 4% of total (14)C under conditions of 5 mM nitrate supply. These results indicate that the decrease in photoassimilate supply to nodules may be involved in the quick and reversible nitrate inhibition of soybean nodule growth.