A simplified enrichment method for selective isolation of Actinomadura rugatobispora strains from soil is described. A. rugatobispora strains poorly produced spores on agar media when 5 to 20 colonies grew on a plate while characteristic spore formation was observed on humic acid-MOPS gellan gum medium. Spores of A. rugatobispora strains were tolerant to dry heating treatment at 120°C for 60 min. A. rugatobispora strains were resistant to 50 μg/ml trimethoprim and 10 μg/ml nalidixic acid, both of which inhibited the growth of nonfilamentous soil bacteria. In addition, these strains were more resistant to 100 μg/ml gentamicin and 20 μg/ml ampicillin than Streptomyces strains, which interfere with the growth of A. rugatobispora selectively. Owing these characteristics, we were successful to isolate 260 strains of this species from 148 out of 547 soil samples (27% of the samples tested). They were recovered from the soil samples with pH values ranging from 5.0 to 8.6, and 91% of the strains were isolated from neutral to slightly alkaline soil (pH 6.0 to 9.0). These results indicated that A. rugatobispora strains were distributed worldwide.
To develop new and improved compounds for future use against drug-resistant bacteria or for chemical modification, it is necessary to continue to screen for new metabolites and evaluate the potential of less known and new bacterial taxa. Selective isolation of rare actinomycetes is one of the major targets of industrial microbiologists in the search for novel compounds with therapeutic activity. Amycolata and Amycolatopsis have also been target for industrial microbiologists in this search. An improved technique, involving the microwave irradiation of soil samples followed by exposure to polyvalent Nocardia/Rhodococcus phages in order to selectively detect the members of the genera Amycolata and Amycolatopsis, has been developed. This improved technique reduced the numbers of colony forming units of unwanted bacteria on isolation plates and hence increased the chances of detecting novel Amycolata and Amycolatopsis species.
DNA fragments recognized and bound by transcriptional factors, AdpA and ArpA, both of which are members in the A-factor regulatory cascade, were isolated. A library of fragmented chromosomal DNA from Streptomyces griseus was constructed either by HaeIII digestion or by sonication, followed by attachment of “catch” linkers at the ends. The library was incubated with a transcriptional activator, AdpA, to allow formation of protein-DNA complexes. The complexes were separated from free DNA by gel mobility shift and the DNA in the complexes was purified by extraction from gel pieces. The purified DNA was then amplified by PCR with the catch linker as primers. For enrichment of the DNA fragments actually bound by the transcriptional factor, the PCR product was subjected to the second and further cycles of the gel mobility shift-PCR procedure. The DNA fragments recovered after cycles of this procedure were cloned in an Escherichia coli plasmid for further characterization. The gel mobility shift-PCR procedure yielded 16 DNA sequences specifically bound by AdpA. This procedure was applied to the isolation of target sequences of another transcriptional factor, ArpA, and two DNA sequences were obtained.
Australia is one of the world’s most biodiverse continents. It has been geologically separated from other continents for over 20 million years, which has allowed a period of extensive evolutionary divergence. As a result, Australia has a very high rate of endemism in both its flora, fauna and associated microflora. Mapping Australian Actinomycete Diversity reveals that Australian substrates contain rare and novel actinomycetes, including novel streptomycetes. There is, therefore, an exiting opportunity within Australia to make contributions in the field of drug discovery, agriculture, food and in the other areas of biotechnology through the acquisition and screening of this microbiota.