High pressure conditions stimulate expression of chloramphenicol acetyltransferase regulated by the lac promoter in Escherichia coli

Abstract Recombinant plasmids with the chloramphenicol acetyltransferase (CAT) structural gene behind several kinds of promoters were tested for expression in Escherichia coli during growth at atmospheric pressure (0.1 MPa) and at high pressure (30 MPa). Expression of the CAT gene from the lac promoter was remarkably activated (approx. 78-fold) by high pressure in the absence of the inducer isopropyl-β-d-thiogalactopyranoside (IPTG). The stimulation of the CAT activity by the lac promoter at high pressure did not simply result from an increased plasmid copy number, because the CAT activities from the other promoters and β-lactamase activities were unaffected at high pressure.     

Phylogeny of symbiotic methanogens in the gut of the termite Reticulitermes speratus

Abstract The phylogeny of a symbiotic methanogen inhabiting the gut of a lower termite, Reticulitermes speratus , was analysed without cultivation. The small subunit ribosomal RNA gene (ssrDNA) and a 640-bp portion of the gene encoding subunit A of methyl coenzyme M reductase ( mcrA ) were amplified from a mixed-population DNA of the termite gut by polymerase chain reaction and cloned. The nucleotide sequence of the ssrDNA and the predicted amino acid sequence of the mcrA product were compared with those of the known methanogens. Both comparisons indicated that the termite symbiotic methanogen belonged to the order Methanobacteriales but was distinct from the known members of this order.     

Comparison of stimulus-response (V-log I) functions in five types of lepidopteran compound eyes (46 species)

Summary Stimulus intensity-response relations (V-log I curves) were electrophysiologically (ERG) determined for the compound eyes of 46 lepidopteran species belonging to five different groups: butterflies (22 species), hesperids (3 species), diurnal sphingids (2 species), diurnal moths (3 species) and nocturnal moths (16 species). The V-log I curves were fitted to the Naka and Rushton equation, in whichn represents the slope of the linear part of each curve. The slopes so determined range fromn=0.35 (the shallowest slope) in nocturnal moths with the greatest dynamic range ton=0.54 (the steepest slope) in diurnal moths andn=0.53 in butterflies both of which have narrow dynamic range. Hesperids (n=0.41) and diurnal sphingids (n=0.38) have intermediate values between butterflies and nocturnal moths.The ratio of rhabdom to retinula volume is significantly higher in nocturnal moths (70–75%), however, those of butterflies and of diurnal moths are very small (2–5%), and hesperids and diurnal sphingids show intermediate ratio (ca. 25%).The slopes of V-log I curves are inversely proportional to the ratio of rhabdom to retinula volume in the various eye types. In all groups except diurnal moths, the light intensities which produce maximal and saturated responses are nearly the same, therefore the nocturnal moths which have the lowest threshold to light increase their sensitivity to dim light mainly by decreasing the slopes of V-log I curves.     

Construction and characterization of the chimeric enzymes between the Bacillus subtilis cellulase and an alkalophilic Bacillus cellulase

The amino acid sequences of cellulase from Bacillus subtilis (BSC) and that from an alkalophilic Bacillus sp. N-4 (NK1) show significant homology in most parts except for the C-terminal portions. Despite the high homology, the pH activity profiles of the two enzymes are quite different; BSC has its optimum pH at 6-6.5, whereas NK1 is active over a broad pH range from 6 to 10.5. In order to identify the structural features which determine such pH activity profiles, chimeric cellulases between BSC and NK1 were constructed using four restriction sites commonly present within the homologous coding sequences, and were produced in Escherichia coli. The chimeric cellulases showed various chromatographic behaviors, reflecting the origins of their C-terminal regions. The pH activity profiles of the chimeric enzymes in the alkaline range could be classified into either the BSC or NK1 type mainly depending on the origins of the fifth C-terminal regions. In the acidic range, the profile was determined only by the origin of the fourth enzyme region from the N terminus. Comparison of the kinetic parameters between pH 5 and 6 using p-nitrophenyl cellobioside as a substrate indicated that the fourth region is responsible for the pH-dependent change of the kcat value. Only a limited number of amino acids in the fourth region may affect on deprotonation of catalytic residues of the cellulases and modulate the catalytic activity in the acidic pH values.     

Isolation and partial characterization of an 87-kilodalton beta-1,3-glucanase from Bacillus circulans IAM1165.

Bacillus circulans IAM1165 produces at least two extracellular beta-1,3-glucanases that lyse fungal cell walls. One of these extracellular enzymes was purified to homogeneity. The molecular mass was 87 kDa, and the pI was 4.3. The optimum temperature of the enzyme reaction was 70 degrees C when laminarin (a soluble beta-1,3-glucan) was used as the substrate. The pH range of the enzyme was broad (pH 4.5 to 9.0), and the optimum pH was 6.5. The enzyme is an endo beta-1,3-glucanase and has a random cleavage pattern.     

Recovery of uranium by immobilized microorganisms

Summary Some attempts were made to recover uranium from sea and fresh water using immobilized Streptomyces viridochromogenes and Chlorella regularis cells. The cells immobilized in polyacrylamide gel have the most favorable features for uranium recovery; high adsorption ability, good mechanical properties, and applicability in a column system. The adsorption of uranium by the immobilized cells is not affected by the pH values between 4 and 9. These results show that uranium adsorption becomes independent of pH after immobilization. The amounts of uranium adsorbed by the immobilized cells increased linearly with temperature, suggesting that the adsorption of uranium by the immobilized cells is an endothermic reaction. The immobilized cells can recover uranium almost quantitatively from both fresh and sea water containing uranium, and almost all uranium adsorbed is desorbed with a solution of Na₂CO₃. Thus the immobilized cells of Streptomyces and Chlorella can be used repeatedly in adsorption-desorption process.Studies on the Accumulation of Heavy Metal Elements in Biological Systems. XXI     

Xylanase Produced by Alkalophilic Bacillus No. C-59-2

Bacillus No. C–59–2 isolated from soil produced a xylanase in alkaline media. The characteristic point of this bacteria was especially good growth in alkaline media, and no growth was observed in neutral media such as nutrient broth. The xylanase of this bacteria was purified by CM-celluIose, hydroxyl apatite and Sephadex G–75 columns. The enzyme was most active at pH 5.5~9 which was much broader and higher than those of other xylanases. The sedimentation constant was about 3.5 S and isoelectric point was pH 6.3. The enzyme was most stable at pH 7 and calcium ion was effective to stabilize the enzyme. The enzyme activity was inhibited by Hg²⁺, Ag²⁺ and Cd^{2 +} Maximum hydrolysis rate of xylan by the enzyme was about 40%. The enzyme split xylan and yielded xylobiose and higher oligosaccharides. Therefore, this enzyme is considered to be a type of endo-xylanase.     

Construction of a chimeric series of Bacillus cyclomaltodextrin glucanotransferases and analysis of the thermal stabilities and pH optima of the enzymes

The cyclomaltodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from the alkalophilic Bacillus sp. strain no. 17-1 was cloned in Escherichia coli. The cloned CGTase gene consisted of a single open reading frame which would encode a polypeptide of 713 amino acids, and the first 27 amino acid residues comprised a signal peptide. The nucleotide sequence and the amino acid sequence of this CGTase (CGTase 17-1) gene had strong homology with those of the CGTase (CGTase 38-2) gene previously cloned in our laboratory from the alkalophilic Bacillus sp. strain no. 38-2, although the enzymic properties of the CGTase 17-1 were distinct from those of the CGTase 38-2. To analyse those enzymic properties further, we constructed 12 chimeric CGTases using three restriction nuclease sites and compared the enzymic properties of the chimeric CGTases. The N-terminal part of the enzyme was important for heat stability, and the pH-activity profile was influenced by both the N- and the C-terminal parts. A third segment was less important for enzymic properties.     

Identification and Growth Characteristics of α-Galactosidase-producing Microorganisms

Two kinds of α-galactosidase-producing microorganisms, strain No. 31–2 and strain No. 7–5, have been isolated from soil and subjected to a determinative study. On the basis of the morphological and physiological characters, the strain No. 31–2 was identified to be belonged to genus Micrococcus and the strain No. 7–5 to genus Bacillus. The former strain, Micrococcus sp. No. 31–2, produced exclusively an intracellular α-galactosidase, and the latter one, Bacillus sp. No. 7–5, secreted the enzyme into culture medium. The cell growth and enzyme production of both strains were observed to reach the maximum under an alkaline culture condition. The intracellular α-galactosidase of Micrococcus sp. No. 31–2 was inducible by galactose, melibiose, and raffinose, while the α-galactosidase of Bacillus sp. No. 7–5 was produced constitutively.