Molecular Identification and Phylogenetic Characterization of Actinomycetes from Libyan Soils via 16S rRNA Gene Sequencing

Abstract

Molecular and genetic analysis of actinomycetes is crucial because these soil bacteria produce the majority of natural antibiotics. This investigation sought to identify actinomycetes using the 16S rRNA gene sequencing technique. Actinomycetes were isolated from soil samples collected near Derna, northeastern Libya. Starch casein medium supplemented with tetracycline and nystatin was employed to isolate actinomycete colonies. Eight isolates were obtained from twenty soil samples collected. DNA was extracted, the 16S rRNA gene was amplified via universal primers 27F/1492R, and the products were sequenced. Sequences were processed using bioinformatics software, compared against the NCBI database, and used to construct a phylogenetic tree in MEGA X via the maximum parsimony method. Five of the eight obtained samples represent sequence identity percentages ranging from 99.28% to 99.63%, which is similar to three registered strains: Streptomyces monomycini ATHUBA 220, Streptomyces olivaceiscleroticus CSSP709, and Streptomyces olivaceiscleroticus JCM 4805. These isolates were successfully deposited in GenBank under accession numbers. One isolate was morphologically similar to Streptomyces but was later characterized as Alterluteimonas muca MHLX1A. The other two isolates were identified as Streptomyces ramulosus NBRC 12812 and Streptomyces monomycini ATHUBA 220. However, their sequence similarity was below 98.5% due to insufficient sequencing data, signifying they may represent novel species. Libyan soils exhibit a high diversity of actinomycetes; consequently, the use of advanced genetic markers – such as DNA-DNA hybridization, multi-locus sequence analysis, and whole-genome sequencing – is recommended for identifying novel strains and searching for their bioactivity.