Masanori Okanishi

Physical characterization of plasmids from Streptomyces kasugaensis MB273.

Abstract Plasmid DNA of molecular weight 6.8 × 106 was isolated from Streptomyces kasugaensis MB273. The plasmid DNA showed a single CsCl-ethidium bromide density gradient centrifugation, in neutral sucrose gradient centrifugation, and in agarose gel electrophoresis. When this DNA was digested with BamHI or SalI endonucleases, an unexpected number of fragments were found on agarose gel electrophoresis. Molecular weight summation of fragments obtained from double restriction enzyme digestions suggested that the plasmid DNA was a mixture of two different plasmids. This was confirmed by constructing recombinant plasmids between S. kasugaensis plasmid DNA and pBR322, and then by isolating two plasmids after SalI endonuclease treatment followed by sucrose gradient centrifugation. One of the plasmids (pSK1) had a single recognition site for BamHI, EcoRI, and SalI, and three sites for BglII. The other plasmid (pSK2) had a single recognition site for EcoRI and BglII, two recognition sites for BamHI, and no cleavage site for SalI. The cleavage maps of these plasmids were constructed using several restriction endonucleases.

Intergenus protoplast fusion between Streptomyces and Micromonospora with reference to the distribution of parental characteristics in the fusants

The protoplasts of two strains of Micromonospora which were sensitive to kanamycin (KM(s)) and utilized raffinose (Raf+), and one strain of Streptomyces griseus which was resistant to KM (KM(r)) and did not utilize raffinose (Raf-), were prepared, mixed in the presence of polyethylene glycol (PEG) and incubated on regeneration agar plates. Recombinant colonies showing KM(r)*Raf+ were obtained at a frequency of 2 x 10(-6). Their recombinants displayed a significant exchange of taxonomic characteristics between the two genera, although the majority appeared similar to the parent Micromonospora in their morphology as well as growth at 40 degrees C. Their patterns of utilization of carbohydrates, amino acids and diammonium hydrogenphosphate were different from those of the Micromonospora. Intermediate or novel types which different from their parents in their tolerance to NaCl and sensitivity to aminoglycoside antibiotics were also observed. Out of the 31 fusants obtained, two showed antimicrobial activity against Bacillus subtilis PCI 219, without any activity against Escherichia coli K-12 or Candida albicans 3147. The active substance may be a newly formed antibiotic, different from streptomycin in S. griseus.