Bunji Maruo

Membrane mutation related to the production of extracellular α-amylase and protease in Bacillus subtilis

Abstract Mutants which had a genetic character to increase the production of both α-amylase and protease simultaneously, were isolated from a transformable strain of Bacillus subtilis Marburg by NTG treatment. This mutation seems to have occurred at a single gene of the bacterial chromosome and was not linked to aro 116 which was closely linked to the α-amylase gene. When this mutation and an α-amylase regulator gene ( amyR h) coexisted in one strain, their synergistic effect on extracellular α-amylase production ws observed. The introduction of this mutation resulted in a loss of competence for the transformation. The SDS disc gel electrophoretic profiles of the membrane proteins from the original strain, the mutants and transformants with this mutation showed a remarkable difference in one component.

Liquid scintillation counting of C14 paper chromatograms

A method is described for the counting of C/sup 14/ biological materials on paper chromatograms. Paper sections are placed in counting vials and are extracted by the solubilizing agent, hyamine. Scintillation solvent is then added, and the samples are counted by a liquid scintillation spectrometer. Absolute radioactivity of each spot can be obtained by any of the following three correction methods. One method involves the dilution of the sample by scintillator solvent, the other uses an internal standard, and the last method involves correction of the observed radioactivity by the counting efficiency obtained from the ratio of counts in two separate windows of the spectrometer. Any of the three methods will give quantitative data if the extraction of the sample from the paper by a solubilizing agent is over 50%. The methods have been applied to the counting of various C/sup 14/ amino acids and C/sup 14/ sugars on paper sections with excellent recovery in radioactivity and reproducibility. (auth)

Ribonucleic acid metabolism in the posterior silkgland of silkworm, Bombyx mori, during the fifth instar

Abstract RNA metabolism in the posterior silkgland of the silkworm, Bombyx mori L, during the fifth instar has been studied. The base composition of the bulk RNA did not change significantly during the instar. 32 P-pulse labeling during the earlier stage of the instar indicated the synthesis of a rapidly turning-over RNA having a base composition different from that of ribosomal RNA and resembling that of DNA. Sedimentation analysis of RNA at this stage revealed the presence of components having different sedimentation constants from those of the ribosomal and s-RNA. At a later stage of the instar, there was no evidence for the occurrence of a rapidly turning-over RNA having a base composition different from that of ribosomal RNA. The role of RNA in silk fibroin synthesis is discussed.

Hybrid α-amylases produced by transformants of Bacillus subtilis. I. Purification and characterization of extracellular α-amylases produced by the parental strains and transformants

Abstract α-Amylase (MAR) produced by a transformable strain of Bacillus subtilis 6160, α-amylase (NAT) from a DNA donor strain of Bacillus natto IAM 1212, α-amylases (NA64 and NA20) from two representative transformants of Bacillus subtilis NA64 and NA20 were purified from the individual culture fluids. The purified α-amylases showed single bands in disc gel electrophoresis. The molecular weights of α-amylases (MAR and NA64) were 55 000, that of α-amylase (NA20) was 42 000 and that of α-amylase (NAT) was 34 000. These four α-amylases belonged to saccharifying type α-amylase such as α-amylase (SAC) produced by Bacillus subtilis var. amylosacchariticus. The general and catalytic properties of α-amylase (MAR) and those of α-amylase(NAT)were different from each other. Almost all properties of α-amylase(NA64) from a transformant were equal to those of α-amylase (MAR) but an elution profile on O-( diethylaminoethyl )- Sephadex A-50 column chromatography of α-amylase (NA64) was rather similar to that of α-amylase (NAT). The properties of α-amylase (NA20) from another transformant were equal to α-amylase (NAT) except the molecular weights. These results suggested that α-amylases (NA64 and NA20) produced by the transformants were hybrid enzymes between the two α-amylases of the parental strains.

Effects of actinomycin D and 5-fluorouracil on the formation of enzymes in Bacillus subtilis

Abstract 1. 1. Effects of actinomycin D and 5-fluorouracil on the formation of α-amylase (EC 3.2.1.1), ribonuclease and alkaline phosphatase (EC 3.1.3.1) in Bacillus subtilis were studied. 2. 2. Actinomycin D, at the concentration of 0.5 μg/ml, inhibited strongly the formation of alkaline phosphatase and α-amylase, but not the formation of ribonuclease. Incorporation of 32P into RNA, and that of [14C]amino acids into protein were strongly inhibited by actinomycin D at the same concentration. 3. 3. 5-Fluorouracil, at the concentration of 100 μg/ml, inhibited the formation of ribonuclease and alkaline phosphatase, but was without any effect on the formation of α-amylase. Incorporation of 32P into RNA, and of [14C]amino acids into protein were slightly inhibited under the same condition. 4. 4. Possible mechanisms underlying these phenomena were discussed taking into consideration some relevant observations made by other workers concerning the modes of action of the two antimetabolites.

Hyperproductivity of extracellular α-amylase by a tunicamycin resistant mutant of Bacillussubtilis

Abstract Several tunicamycin resistant mutants were obtained from Bacillus subtilis NA64. One of them, B7 strain produced a 5-fold larger amount of α-amylase than NA64 did. Only the amount of α-amylase, among excreted proteins, was enhanced. Genetic analyses by transformation suggested that a single mutation in B7 induced both resistance to tunicamycin and hyperproductivity of extracellular α-amylase.

Hybrid α-amylases produced by transformants of Bacillus subtilis. II. Immunological and chemical properties of α-amylases produced by the parental strains and the transformants

Abstract Six α-amylases (MAR, NAT, NA20, NA64, SAC and LIQ) produced by a transformable strain of Bacillus subtilis 6160, by a DNA donor strain of Bacillus natto IAM 1212, by representative transformants Bacillus subtilis NA20 and NA64, by Bacillus subtilis var. amylosacchariticus and Bacillus amyloliquefaciens F were compared with one another with regard to their immunological and chemical properties, including peptide mappings. Five α-amylases (MAR, NAT, NA20, NA64 and SAC) cross-reacted almost equally with rabbit anti α-amylase (SAC) serum but not with rabbit anti α-amylase (LIQ) serum, α-Amylases (MAR and NA64) contained about 9% of carbohydrate but not the others. The different indices calculated from the amino acid compositions showed not only structural similarity among the five α-amylases but also considerable difference between them. More than 80% of the tryptic peptide components from α-amylases (MAR and NA64) seemed to be identical and 4 or 5 specific peptides for each were detected. About 88% of the tryptic peptides from α-amylase (NAT) seemed to be in common with those from α-amylase (NA20). The latter contained more specific peptides than the former. The peptide maps of the five saccharifying type α-amylases (MAR, NAT, NA20, NA64 and SAC) showed that at least 12 peptides seemed to be common.

Purification and properties of a cross-reacting material related to α-amylase and biochemical comparison with the parent α-amylase

1. 1. A cross-reacting material serologically related to α-amylase was purified from the culture medium of Bacillus subtilis MO7 by means of (NH4)2SO4 precipitation, CM-cellulose, Sephadex G-75 and DEAE-Sephadex A-50 column chromatography and finally preparative disc gel electrophoresis, and compared with the α-amylase (NA-64) produced by the parental strain of B. subtilis NA-64. 2. 2. The purified cross-reacting material showed a single band in analytical disc gel electrophoresis and was entirely free from amylase, protease, ribonuclease, alkaline phosphatase, β-galactosidase, α- and β-glucosidase activity. 3. 3. The cross-reacting material and α-amylase (NA-64) showed almost the same molecular weight, pH- and thermo-stability, mobility on electrophoresis, sensitivity to EDTA and p-mercuribenzoate, and immunological reactivity against rabbit anti-α-amylase sera. 4. 4. The preparation contains about 6% glucosamine and about 8% neutral carbohydrate; the α-amylase (NA-64) contains about 7% glucosamine and about 2% neutral carbohydrate. 5. 5. Differences between the cross-reacting material and α-amylase (NA-64) were found in the elution profiles obtained on gel filtration with Sephadex G-75 columns, and in the peptide maps obtained by two-dimensional chromatography on cellulose thin-layer plates. One specific peptide from the cross-reacting material and two specific peptides from α-amylase (NA-64) were observed. 6. 6. Preliminary genetic studies indicate that the cross-reacting material-forming mutant has a small “inframe” deletion (or inversion).

The binding of the parental DNA from bacteriophages to the cell membrane of Escherichia coli

Abstract After infection of Escherichia coli with phages λ, Φ80, 434 or P1, about 50–60 % of the parental DNA was bound to a fast-sedimenting fraction as shown by sucrose gradient analysis. The cell component binding the phage DNA was tentatively identified as cell membrane. When the cell infected with λ vir at a high multiplicity was superinfected with 434, Φ80 or P1, the binding of 434 DNA to the cell membrane was inhibited, but that of Φ80 DNA or P1 DNA was not. These results suggest that the binding sites for λ DNA are identical with those for 434 DNA but different from those for Φ80 DNA or P1 DNA. When the λ vir infected cell was superinfected with λ–Φ80 hybrid phages, Hyb68 or i 80 h λ , the binding of Hyb68 DNA which contains the right half (int-R) of λ DNA was inhibited, but that of i 80 h λ DNA which lacks the right half of λ DNA was not. These results suggest that the function required for the “recognition” of the λ specific binding sites is localized in the right half of λ DNA.

Stimulation of amylase formation by an amine from Bacillus subtilis

An active factor which stimulates amylase formation by Bacillus subtilis has been extracted from B. subtilis cells and purified. The factor resembles alkyldiamines or polyalkylamines in its chromatographic behavior, its reaction with ribonucleic acids and other types of reactions. Authentic samples of amines such as putrescine, cadaverine, spermidine and spermine have been found to stimulate amylase formation at high concentrations. None of them however is identical with the factor. The factor also stimulates the production of other exo-enzymes of B. subtilis, e.g., proteases, ribonuclease, and γ-glutamyltransferase. Moreover it stimulates the turnover of RNA and polyphosphate in the cells to a greater extent than it stimulates enzyme production. In vitro experiments showed that in the presence of the factor, RNA associates to larger molecules than those of the original RNA. The chemical structure of the factor and its biological functions are discussed.