Identification of Novel Antimicrobial Actinomycetes

Genomic DNA was extracted from four actinomycetal strains that showed the best antimicrobial activity according to the method described by Hong et al. [64 (link)]. The 16S rRNA gene was amplified with a set of bacteria-universal primers (Invitrogen, USA); the primers 27F (5-GAGTTTGATCCTGGCTCA-3) and 1498R (5-ACGGCTACCTTGTTACGACTT-3), which are complementary to the conserved regions at the 5- and 3- ends of the E. coli 16S rRNA gene [65 (link)]; 3 mM MgCl₂; 3 mM dNTPs; 5 µL of Taq buffer; and 1 U Taq DNA polymerase (Invitrogen, Waltham, MA, USA). PCR amplification was performed on a cycler PCR machine (Bio-Rad Laboratories, Segrate, Italy), with the initial denaturation at 94 °C for 5 min, followed by 50 cycles of amplification (94 °C for 1 min, 54 °C for 1 min, and 72 °C for 2 min) and an extension step (72 °C for 5 min). Of each PCR product, 50 ng/μL was used to prepare the samples, which were delivered to MacroGen Company in Korea (http://www.dna.macrogen.com, accessed on 20 October 2021) following their specifications. The sequences were analyzed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST, accessed on 20 October 2021) to preliminarily identify the strains. The cluster analysis was performed using the MEGAX (10.1.8) software package.