Biosystematics of the genus Dactylosporangium and some other filamentous actinomycetes

PhD ThesisThis study tested the hypothesis that a relationship exists between taxonomic diversity and antibiotic resistance patterns of filamentous actinomycetes. To this end, 200 filamentous actinomycetes were selectively isolated from a hay meadow soil and assigned to groups based on pigments formed on oatmeal and peptone-yeast extract-iron agars. Forty-four representatives of the colour-groups were assigned to the genera Dactylosporangium, Micromonospora and Streptomyces based on complete 16S rRNA gene sequence analyses. In general, the position of these isolates in the phylogenetic trees correlated with corresponding antibiotic resistance patterns. A significant correlation was found between phylogenetic trees based on 16S rRNA gene and vanHAX gene cluster sequences of nine vancomycin-resistant Streptomyces isolates. These findings provide tangible evidence that antibiotic resistance patterns of filamentous actinomycetes contain information which can be used to design novel media for the selective isolation of rare and uncommon, commercially significant actinomycetes, such as those belonging to the genus Dactylosporangium, a member of the family Micromonosporaceae. A culture-independent, nested PCR procedure based on genus-specific oligonucleotide primers detected the presence of Dactylosporangium strains in 14 out of 21 environmental samples. Clones generated from the 14 positive samples formed novel phyletic lines in the Dactylosporangium 16S rRNA gene tree. Presumptive dactylosporangiae were isolated from 7 of these samples using a medium designed to be selective for members of the genus Dactylosporangium. One hundred and two out of 219 representative presumptive dactylosporangiae were considered to be bona fide members of the genus Dactylosporangium as they gave PCR amplification products with primers specific for this taxon. Representatives of the Dactylsporangium isolates formed distinct phyletic lines in the Dactylosporangium 16S rRNA gene tree were designated as new species, namely Dactylosporangium luridum sp. nov. and Dactylosporangium luteum sp. nov., based on a polyphasic study. Similarly, “Dactylosporangium salmoneum” NRRL B-16294 was validly described as a new species, Dactylosporangium salmoneum sp. nov., nom. rev. In addition, “Dactylosporangium variesporum” NRRL B-16296 was transferred to the genus Saccharothrix as Saccharothrix variisporea corrig. (ex. Tomita et al. 1977) sp. nov., nom. rev. Some of the representative Dactylosporangium isolates inhibited the growth of Bacillus subtilis, Kocuria rhyzophila and Staphylococcus aureus strains, suggesting that novel Dactylosporangium strains might be a rich source of novel antibiotics. Verrucosispora maris AB-18-032, another member of the family Micromonosporaceae, produces atrop-abyssomicin C, the first natural inhibitor of the para-aminobenzoic acid pathway. The self-protective mechanism of this strain was sought by conjugating an atrop-abyssomicin C sensitive Streptomyces griseus strain against a genomic DNA library prepared from V. maris AB-18-032. Seven resultant resistant exconjugants were screened for atrop-abyssomicin C resistance genes using four designed PCR primers. The failure to detect PCR amplification products suggests that the resistance shown by the exconjugants is conferred by mutation within the S. griseus strain or by cloning of unidentified resistance genes from the V. maris strain.National Institute for International Education of the Korean Government (NIIED) Newcastle Universit