Isolation, characterization, identification, and investigation of secondary metabolites of streptomyces bacteria from extreme soils of Kars and Iğdir (Türkiye)

This thesis aimed to isolate Streptomyces species producing antibacterial metabolites from the extreme soil habitats of the hypersaline Iğdır/Tuzluca and the cryophilic Kars/Sarıkamış regions. In addition to the morphological, biochemical, and molecular characterization of the isolates, the study focused on the extraction and characterization of secondary metabolites demonstrating potent antibacterial activity. Initially, the physicochemical properties of soil samples from the specified extreme habitats were determined, and putative Streptomyces isolates were obtained using serial dilution combined with spread and pour plate techniques. The antibacterial activities of the isolates against test bacteria were evaluated. Based on their activity spectra, the morphological and biochemical properties of the selected isolates were determined. For molecular identification, the 16S rRNA, rpoB, and recA gene regions were sequenced to perform phylogenetic analyses. Subsequently, fermentation conditions were optimized for the most promising isolate to determine the optimal medium and incubation time for metabolite production. Finally, the obtained metabolite was purified via chromatographic methods, and ¹H-NMR and FT-IR analyses were carried out in order to elucidate its structural properties. As a result of the study, 34 Streptomyces isolates were recovered, among which BH006, BH011, BH022, BH026, and BH034 exhibited high antibacterial activity using the cross-streak method. These isolates produced inhibition zones measuring: for BH006, 24 mm against Staphylococcus aureus, 35 mm against Bacillus cereus, and 24 mm against Escherichia coli; for BH011, 15 mm against B. cereus; for BH022, 13 mm against S. aureus, 25 mm against B. cereus, and 16 mm against E. coli; for BH026, 39 mm against S. aureus, 40 mm against B. cereus, and 37 mm against E. coli; and for BH034, 18 mm against S. aureus, 17 mm against B. cereus, 11 mm against Enterococcus faecalis, and 13 mm against E. coli. Based on morphological and biochemical tests, these five isolates were determined to display distinct species-level characteristics. Phylogenetic analyses revealed that isolates BH006, BH011, BH022, BH026, and BH034 were closely related to Streptomyces ficellus, Streptomyces rochei, Streptomyces bikiniensis, Streptomyces gobitricini, and Streptomyces inhibens, respectively. Fermentation experiments demonstrated that the metabolites of isolate BH026 exhibited the highest antibacterial activity against S. aureus, B. cereus, E. faecalis, and E. coli. For this isolate, GYM broth (glucose, yeast extract, and malt extract) was identified as the optimal production medium, with maximal metabolite production observed on the 3rd day. However, despite purification efforts, the chemical structure of the active metabolite could not be fully elucidated due to insufficient yield. Furthermore, a metagenomic analysis was conducted on four selected soil samples to investigate microbial diversity and situate the isolated Streptomyces species within their natural microbial community. To this end, total genomic DNA extracted from the soil samples was sequenced using next-generation sequencing (NGS), followed by taxonomic classification via bioinformatic analyses. The analysis revealed extensive diversity, comprising 22 phyla, 38 classes, 91 orders, 186 families, 364 genera, and 662 species. Although the generic composition varied across the samples, Pasteurella and Sphingomonas were identified as the dominant genera in all soil samples.