Vpliv zaznavanja kvoruma na sintezo skupnih dobrin pri bakteriji Bacillus subtilis

Skočir, Erika (author), Danevčič, Tjaša (mentor)

Bakterija Bacillus subtilis je znana po tem, da je sposobna iz celice izločati velike količine različnih produktov kot skupnih dobrin. V nalogi nas je zanimalo, če je izločanje teh skupnih dobrin regulirano s sistemom za zaznavanje kvoruma (QS), kot je to pri nekaterih drugih bakterijskih vrstah. Kot primer skupne dobrine smo vzeli zunajcelične proteaze, ki so enostavne za merjenje in dobro preučene, saj se jih industijsko že veliko časa pridobiva s pomočjo določenih vrst iz rodu Bacillus. Med seboj smo primerjali divji tip naravnega izolata ter dve mutanti, ki imata prekinjena gena comQ in degQ. Prvi sodeluje pri procesiranju signalov za zaznavanje kvoruma, drugi pri sintezi zunajceličnih proteaz. Najprej smo preizkusili različna gojišča, ki so vsebovala različne kombinacije virov hranil, in primerjali hitrost rasti, sposobnost sinteze in izločanja zunajceličnih proteaz ter proteazno aktivnost. Nato smo med seboj primerjali vse tri seve v proteinskih gojiščih in preverili, kako se spreminjata hitrost rasti in koncentracija zunajceličnih proteaz glede na čas inkubacije in glede na okvare genov. Vsem trem sevom smo označili promotor gena za zunajcelično proteazo subtilizin (AprE) z zelenim fluorescenčnim proteinom GFP, da smo spremljali ne samo koncentracijo končnega produkta, ampak tudi prepisovanje genov. Kulturam označenih in neoznačenih sevov gojenih v gojišču MSgg smo merili intenziteto fluorescence s fluorimetrom. Izkazalo se je, da je imel divji tip v vseh preizkušenih gojiščih boljšo rast in je sintetiziral večje količine zunajceličnih proteaz kot mutante, torej je bila proteazna aktivnost divjega tipa vedno največja. Regulacijo sinteze zunajceličnih proteaz, kot primer skupnih dobrin, lahko zato tudi pri bakteriji B. subtilis povežemo z zaznavanjem kvoruma.

• Language: Slovenian
• Keywords: zaznavanje kvoruma, skupne dobrine, zunajcelične proteaze, Bacillus subtilis, proteazna aktivnost
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [E. Skočir]
• Year: 2017
• PID: 20.500.12556/RUL-99152
• UDC: 579.22/.26:579.852.1:577.15
• COBISS.SI-ID: 4868216
• Publication date in RUL: 24.12.2017

Secondary language

• Language: English
• Title: Impact of the quorum sensing on the public goods synthesis in Bacillus subtilis

Abstract

Bacillus subtilis is a bacterium that is well known for its ability to secrete large amounts of its products as public goods. We were interested if the secretion of these public goods is regulated by quorum sensing (QS) system as it is in some other bacterial species. Extracellular proteases were used as an example of public goods, because they are easy to quantify and are also very well studied due to the use of Bacillus genus as an industrial producer of proteases for many years. The study was made by comparing wild type isolate and two mutant strains with mutations in comQ and degQ genes. ComQ is involved in QS signal processing, while DegQ is responsible for extracellular protease production. Firstly, we compared different growth media that contained different combinations of nutrients. After 24 hours of incubation, a comparison of growth rate and the ability to synthesize and excrete proteases was made. From those results the protease activity was calculated. Then we compared all three strains growing them in protein media and followed the growth and the concentration of extracellular proteases trough incubation time. We wanted to see if there was a difference between mutants and the wild type strain. The promoter of the aprE gene for subtilizin – an important extracellular protease was then fused with green fluorescent protein GFP to follow the transcription process. We measured the fluorescence intensity with fluorimeter for marked and unmarked strains, growing in liquid MSgg medium. The results show a better growth and higher extracellular protease production for wild type isolate in all tested growth media so the protease activity was always the highest for wild type strain. Extracellular protease production as an example of public goods regulation in B. subtilis can therefore be linked to it's quorum sensing system.

• Keywords: quorum sensing, public goods, extracellular proteases, Bacillus subtilis, protease activity