Molecular characterization of Streptomyces was carried out by 16S rRNA analysis. The cells from the biomass were harvested by centrifugation, washed, and re-suspended in TE buffer (10 mM Tris/HCl, 1 mM EDTA; pH 8.0). Genomic DNA was extracted by a standard protocol and the PCR amplification was conducted in Eppendorf Master Cycle gradient AG22331 (Model No. 5331), using appropriate forward primer 27F (5' AGA GTT TGA TCC TGG CTC AG 3') and reverse primer 1525R (5' AGA AAG GAG GTG ATC CAG CC 3') in the E. coli numbering system [8 (link), 9 ]. The amplified products were sequenced in Applied Biosystem Sequencer (ABI 3730XL DNA Analyzer) with appropriate primers.
This 16S rRNA gene sequence was used for phylogenetic analysis. The 16S rRNA gene sequence related taxa were acquired from the GenBank database and the primer was designed using the Primer 3 software. Multiple sequence alignment was conducted using the Clustal W program and the phylogenetic tree was constructed using the neighbor-joining method [10 (link), 11 (link)] using the MEGA7 software (https://www.megasoftware.net/) and phylogeny program (http://www.phylogeny.fr/). The evolutionary distances for the neighbor-joining and maximum likelihood tree were calculated by Kimura’s two-parameters method. The topologies of each tree have been evaluated using bootstrap resampling methods based on 1,000 replications.