Streptomyces virginiae is one of the most difficult Streptomyces strains to transform because of its strong restriction barrier. We succeeded in transforming S. virginiae by combining heat treatment of protoplasts and in vitro methylation of plasmids. In order to avoid the host restriction barrier, plasmids were modified by methylase AluI, and S. virginiae protoplasts were treated at 42°C for 15 min prior to transformation. Under the optimal conditions, plasmids pIJ486, pKC1064 and pUWL-KS isolated from Streptomyces lividans were introduced into S. virginiae at a frequency of 1.4×103, 7.0×102 and 1.2×103 transformants per μg DNA, respectively. Plasmid pKC1064 and pUWL-KS isolated from Escherichia coli DH5α could not transform S. virginiae, but those isolated from E. coli JM110 (dam, dcm) could transform at a frequency of 1.0×102 and 2.4×102 transformants per μg DNA, indicating the presence of a methylation-specific restriction system in S. virginiae.
A total of 93 soil samples were collected at 3 sites in the southern, at 4 sites in the northwestern, and at 2 sites in the northeastern regions of the Republic of South Africa. When these samples were subjected to the conventional dilution plate method for the isolation of actinomycetes, the genus Streptomyces was found to be dominant, and of 135 total isolates, 53 (39%) were rare actinomycetes. However, the newly developed high-speed centrifugation method enabled us to isolate 335 isolates, of which 295 (88%) were rare actinomycete strains. These included the rare genera Planomonospora and Planobispora, which have been known to have a very low population density in soils. These two genera were detected only in soil samples collected from the extremely arid area, called “Namaqualand”, in the northwestern region of the Republic of South Africa.
A DNA fragment, which suppressed formation of mucoidal colonies, was isolated from Rhodococcus rhodochrous rough strain R-2. This fragment contained two ORFs in the same orientation, and their deduced amino acid sequences showed no significant homology to reported proteins. Essential region for suppression of mucoidal morphology was determined by subsequent subcloning.
We examined the propeptin production in 10 species of mesophilic Microbispora. Three strains which have closely DNA homology to Microbispora rosea, M. rosea (IFO 14044), M. rosea subsp. nonnitritogenes (IFO 14045) and M. indica (IFO 14879) produced propeptin, other 7 strains did not. The results suggested some relationship between secondary metabolite production and taxonomy.
Main studies on Streptomyces, especially S. griseus, during about thirty years of the author’s career are outlined. 1. Studies on streptomycin biosynthesis using resting mycelium-suspension culture of S. griseus revealed the following; 1) Streptomycin 6-phosphate is the final precursor, which is converted to streptomycin by a specific alkaline phosphatase, 2) Streptomycin 6- phosphotransferase contributes to self-protection from a toxic effect of streptomycin, and 3) Suppression of the phospholipid cycle of cell wall synthesis results in the accumulation of glucosamine 6-phosphate in the resting mycelium and the enhancement of streptomycin production 2. Genetic studies provided the following; 1) S. griseus has two genes contributing to self-protection against streptomycin, 2) Pleiotropic mutants were obtained at a high frequency by incubating mycelium under stress condition for growth, and 3) Physical macrorestriction map of S. griseus 2247 strain was constructed.