Characterization of active prophages from microscale Bacillus subtilis soil isolatesPolajnar, Mercedes (Avtor) Dragoš, Anna Magdalena (Mentor) Štefanič, Polonca (Komentor) prophagesBacillus subtilissoil isolatesprophage inductionkin discriminationThis master’s thesis investigates the presence, diversity and activity of prophages in microscale Bacillus subtilis PS soil isolates. Prophages are viral genomes integrated into bacterial chromosomes that can remain dormant or be induced to produce phage particles. Their activation affects bacterial physiology, gene exchange and competition, yet their role in natural B. subtilis isolates remains poorly understood. To address this, 40 sympatric PS isolates were experimentally screened for phage activity, titers and variety of plaques. Prophage induction was carried out both spontaneously (SPI) and chemically with mitomycin C (MitC). Filtered supernatants were analyzed for phages using spotting and confluent plaque assays on the prophage-free indicator strain B. subtilis Δ6. This was followed by phage isolation from plaques, enrichment on liquid and solid medium, precipitation, DNA extraction, PCR (plaque and genomic PCR) and transmission electron microscopy (TEM). A swarm boundary assay was also performed to test whether phage release occurs during social interactions. Active prophages were detected in six strains (PS-11, PS-24, PS-25, PS-233, PS-237, PS-261), which is 15% of all isolates. The plaques showed a broad range of morphologies, including large ringed plaques, numerous small plaques, and two different plaque types from one supernatant. MitC was consistently more effective than SPI, producing higher titers. In some samples plaques co-occurred with inhibition zones; overall 31 strains showed antimicrobial activity. Enrichment allowed successful phage DNA extraction, TEM of PS-11 lysates revealed Podovirus-like and Siphovirus-like phage morphologies and most PCRs confirmed phage regions. Swarming conditions did not reveal detectable phage plaques, although inhibition zones were present, indicating antimicrobial release. Together, these findings confirm that inducible prophages are present and diverse among PS isolates, and they emphasize the complex roles prophages may play in the ecology, evolution, and fitness of B. subtilis. This work provides a base for future studies on prophage activity in natural settings, with the potential to further investigate how these elements shape bacterial interactions and competitive dynamics.20252025-10-11 07:15:08Magistrsko delo/naloga174974sl