Characterization of active prophages from microscale Bacillus subtilis soil isolates

Polajnar, Mercedes (author), Dragoš, Anna Magdalena (mentor), Štefanič, Polonca (comentor)

This 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.

• Language: English
• Keywords: prophages, Bacillus subtilis, soil isolates, prophage induction, kin discrimination
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Year: 2025
• PID: 20.500.12556/RUL-174974
• COBISS.SI-ID: 252864515
• Publication date in RUL: 11.10.2025

Secondary language

• Language: Slovenian
• Title: Karakterizacija aktivnih profagov izolatov Bacillus subtilis iz talnega mikrohabitata

Abstract

To magistrsko delo preučuje prisotnost, raznolikost in aktivnost profagov v PS izolatih Bacillus subtilis iz talnega mikrohabitata. Profagi so virusni genomi, integrirani v bakterijske kromosome, ki lahko ostanejo v mirovanju ali pa so inducirani in tvorijo fagne delce. Njihova aktivacija vpliva na fiziologijo bakterij, izmenjavo genov in tekmovanje, vendar je njihova vloga v naravnih izolatih B. subtilis še vedno slabo raziskana. Z namenom preučitve aktivnosti fagov in raznolikosti plakov ter pridobivanja fagnih titrov je bilo eksperimentalno testiranih 40 simpatričnih PS izolatov. Profagna indukcija je bila izvedena spontano (SPI) in kemično z mitomicinom C (MitC). Filtrirani supernatanti so bili analizirani za prisotnost fagov prek izvedbe testa plakov na indikatorskem sevu B. subtilis Δ6. Sledila je izolacija fagov iz plakov, obogatitev fagov v tekočem in trdnem mediju, precipitacija fagov, izolacija fagne DNA, PCR (PCR iz plakov in genomski PCR) ter transmisijska elektronska mikroskopija (TEM). Izveden je bil tudi test rojenja na B mediju za preverjanje spontanega sproščanja fagov med socialnimi interakcijami. Aktivni profagi so bili zaznani v šestih sevih (PS-11, PS-24, PS-25, PS-233, PS-237, PS-261), kar predstavlja 15 % vseh izolatov. Morfologija plakov je bila zelo raznolika, vključno z velikimi obročastimi plaki, številčnimi majhnimi plaki in dvema različnima tipoma plakov iz enega supernatanta. MitC je bil učinkovitejši kot SPI in je povzročil višje titre. V nekaterih primerih so se plaki pojavljali skupaj s conami inhibicije; skupno je 31 sevov pokazalo protimikrobno aktivnost. Obogatitev je omogočila uspešno izolacijo fagne DNA, TEM lizatov PS-11 je razkril morfologiji fagov, podobni tipoma Podovirus in Siphovirus, večina PCR analiz pa je potrdila fagne regije. Rezultati rojenja niso pokazali fagnih plakov, prisotne pa so bile cone inhibicije kot posledica protimikrobnih spojin. Rezultati potrjujejo, da so raznoliki inducibilni profagi prisotni v PS izolatih, in poudarjajo kompleksne vloge profagov v ekologiji, evoluciji in prilagoditvi B. subtilis. To delo predstavlja osnovo za prihodnje raziskave profagne aktivnosti v naravi ter omogoča nadaljnje preučevanje, kako ti elementi oblikujejo bakterijske interakcije in dinamiko tekmovanja.

• Keywords: profagi, Bacillus subtilis, talni izolati, profagna indukcija, sorodstvena diskriminacija