Biodiversity of uncultured Streptomyces involved in bioactive substance production from soil in Thailand

Abstract

Streptomyces, gram-positive filamentous bacteria and G-C rich nucleotide, have capability of producing several bioactive compounds and predominantly exhibit in soil. This study is aimed to investigate the genetic diversity of Streptomyces in soil samples in Thailand by using culture-independent method with total genomic DNA extraction and purification from soil. The 16S rDNA was then amplified by nested Polymerase Chain Reaction (nested-PCR) technique using universal primers and Streptomyces specific primers for the first and second PCR reactions respectively. The PCR product about 1 kb was obtained and cloned into T/A cloning vector to make clone library. The biodiversity of 16S rRNA genes was determined by Restriction Fragment Length Polymorphism (RFLP). Based on in silico restriction endonuclease digestion of seventy-nine 16S rDNA sequence data [Streptomyces (70 species), Actinomyces (6 species), and outgroup bacteria (3 species)] derived from public sequences databases (RDP and NCBI) using NEBcutter program with 33 restriction endonucleases and calculation of restriction distance using Nei-Li method and construct of Neighbor-joining tree using PAUP program various tpes of RFLP patterns were elucidated. It was found that a number of OTUs of RFLP patterns derived from MspI (isoschizomer of HpaII) yielded high level of average restriction site per species (10.50), and good correlation between the phylogenetic distribution and the production of bioactive compounds. The MspI enzyme was, therefore, selected for the analysis of 16S rDNA gene isolate and amplified from soil in different locations of Thailand (mountain, mangrove forest, and paddy field) in order to investigate the biodiversity of such Streptomyces as describe previously. The results showed that 100 clones of 16S rDNA of such soils revealed RFLP patterns of 16 OTUs. Comparison of the RFLP patterns from soils to simulated RFLP patterns (digested with MspI) showed that dominant OTU type was ‘a’ type. The ‘a’ type of RFLP pattern contains several important bioactive producing Strepomyces such as S. venezuelae and S. fridae which are antibiotic producers. Three OTUs (i, m, and f type) were found to match S. lavendulae (growth promotant), Micromonospora olivasterospora (antibacterial) , and Thermomonospora chromogena, respectively.