Stabilnost izmenjave DNA v tekočih kulturah in v biofilmih bakterije Bacillus subtilis

Ruperčič, Maja (author), Mandić Mulec, Ines (mentor), Dragoš, Anna Magdalena (comentor)

Bacillus subtilis je naravno kompetentna bakterija, ki je sposobna tvoriti biofilm. Zadnje raziskave kažejo, da je horizontalni genski prenos znotraj statične kulture uspešnejši, kot v planktonski kulturi. V magistrskem delu smo kvantificirali izmenjavo DNA v statični (biofilmu) in planktonski (tekoči) kokulturi sevov B. subtilis, sev A odporen proti antibiotiku spektinomicinu (A(spec)) in sev B proti antibiotiku kloramfenikolu (B(cat)). Sposobnost sprejema DNA, rast, morfologija in biomasa biofilma obeh sevov je bila primerljiva. Ugotovili smo, da se v kokulturi sevov A(spec) in B(cat), ki ju gojimo v tekočem gojišču MSgg, transformante pojavijo že po 4 urah inkubacije, vendar je frekvenca transformacije nizka. Frekvenca transformacije statične in stresane kulture je bila signifikantno različna (p=0,002). Izmenjava DNA je bila višja v statični kot v stresani kulturi po 12-ih in 24-urah inkubacije (p12 ur=0,113; p24 ur =0,013). Poleg tega smo merili tudi izražanje kompetenčnih genov v gojišču MSgg pri sevu B. subtilis PS-216 wtGFP (amyE::RFP comGA-gfp), ki konstitutivno izraža RFP ter GFP pod kontrolo promotorja operona comGA. Ugotovili smo, da kulturi dosežeta maksimum izražanja comGA-gfp po šestih urah inkubacije. Ob dodatku DNAze v gojišče smo pokazali, da ta ne vpliva na izmerjeno koncetracijo eDNA v kokulturi. Čeprav je bila izmenjava DNA med sevi višja v statični kulturi, pa naši rezultati kažejo, da izmenjava poteče verjetno še preden celice tvorijo biofilm in zato na podlagi naših rezultatov ne moremo zaključiti, da je izmenjava DNA pri B. subtilis učinkovitejša v biofilmu.

• Language: Slovenian
• Keywords: Biotehnologija, Bacillus subtilis, kompetenca, biofilm, planktonska kultura
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [M. Ruperčič]
• Year: 2017
• PID: 20.500.12556/RUL-92403
• UDC: 602:3:579.852.11:579.23/.25(043.2)
• COBISS.SI-ID: 8735609
• Publication date in RUL: 03.06.2017

Secondary language

• Language: English
• Title: Stability of dna-exchange in planktonic and biofilm lifestyle of Bacillus subtilis

Abstract

Bacillus subtilis is a natural competent bacteria capable of biofilm formation. Recent studies have uncovered higher efficiency of horizontal gene transfer in static culture than in planktonic culture. The aim of our study was to quantify and compare the DNA exchange in biofilm or in planktonic coculture. First we tested wether the model strains A(spec) and B(cat) are good candidates to perform DNA exchange assays. Results indicated that their growth dynamics, biofilm morphology, final biomass yield and the ability to take up exogenous DNA were similar and comparable to the parental strain B. subtilis PS-216. Thus we concluded that both strains are suitable to perform DNA exchange. The first transformed cells have been already detected after just four hours of cocultivation and the transformation frequency was significantly higher in static (biofilm) than in planktonic coculture after 12 and 24 hour of incubation (p12h=0,113; p24h =0,013). However, the overall frequency of transformation was rather low in MSgg medium. The expression of competence genes was monitored in time using reporter strain PS-216 wtGFP (amyE::RFP comGA-gfp). The RFP protein was constitutively expressed and the GFP protein was under the control of the comGA promoter. We observed that cocultivated strains reach expression maximum after 6 hours of incubation which is before the biofilm forms. Interestingly, addition of DNAse to co-culture did not affect the concentration of eDNA nor was the eDNA concentraion different in two modes of growth. Although we showed that DNA exchange was higher in static than in planktonic culture we cannot conclude that DNA exchange is more effcient in biofilms compared to the planktonic culture as the the transformation maximum was way in advance biofilms formed on the liquid-air interface.

• Keywords: Biotechnology, Bacillus subtilis, competence, biofilm, planktonic culture