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Bacillus subtilis živi v okolju, kjer potekajo neprestane osmotske spremembe. V magistrski nalogi smo želeli ugotoviti vpliv sistema za zaznavo celične gostote oziroma kvoruma ComQXPA in z DegQ uravnanega sistema DegS-DegU na prilagajanje bakterije B. subtilis na povišano slanost. Vključili smo divji tip seva PS-216, mutanto PS-216 ΔcomQ, ki ne tvori aktivnega signala ComX sistema ComQXPA, in mutanto PS-216 ΔdegQ, ki ne tvori DegQ. Sevom smo določili rast in morfološke lastnosti kolonij v tekočih in na trdnih gojiščih SM in MSgg z različnimi koncentracijami NaCl. V nadaljevanju smo spremljali izražanje fluorescenčno označenih promotorjev genov proH, epsA in srfAA pri različnih slanostih tekočega gojišča MSgg. Primerljive velikosti površin nastalih kolonij pri mutanti ΔcomQ pri vseh slanostih in hitrejši začetek izražanja promotorja gena proH ob povišani slanosti v primerjavi z divjim tipom kažejo na boljšo prilagoditev mutante ΔcomQ na povišano slanost. Izražanje promotorja gena epsA je bilo pri mutanti ΔcomQ višje kot pri divjem tipu, kar kaže na boljšo tvorbo biofilma. Manjša aktivnost sistema DegS-DegU pri mutanti ΔdegQ vpliva na morfologijo kolonij, medtem ko je dinamika začetka tvorbe kolonij in njihova končna površina podobna kot pri divjem tipu. Promotorja genov proH in epsA se izražata pozneje in manj intenzivno, kar kaže na slabšo prilagoditev na povišano slanost ter inhibirano tvorbo biofilma. Izražanje promotorja gena srfAA je bilo najvišje pri mutanti ΔdegQ, sledil je divji tip in najnižjo aktivnost smo zasledili pri mutanti ΔcomQ. Iz pridobljenih rezultatov lahko sklepamo, da odsotnost sistema za zaznavanje kvoruma ComQXPA pospeši prilagoditev mutante ΔcomQ na povišano slanost, medtem ko manjša aktivnost sistema DegS-DegU prilagoditev mutante ΔdegQ na povišano slanost upočasni. Bacillus subtilis lives in an environment with frequent osmotic changes. In this master's thesis we aimed to investigate the influence of the DegQ-controlled DegS-DegU system and ComQXPA system, involved in cell density or quorum sensing, on the adaptation of B. subtilis to high salinity. Strains included were PS-216 wild-type (wt) strain, mutant strain PS-216 ΔcomQ without production of an active signal ComX of the ComQXPA system, and mutant strain PS-216 ΔdegQ without DegQ production. Growth and morphological properties of colonies in liquid and solid SM and MSgg media with different NaCl concentrations were determined. In addition, the expression of fluorescently labelled proH, epsA, and srfAA gene promoters was monitored at different salt levels in MSgg liquid medium. Similar areas of the colonies of the mutant ΔcomQ at all salinities and faster beginning of the proH gene promoter expression at increased salinity in comparison to the wt strain show more efficient mechanism of adaptation of the mutant ΔcomQ strain to high salinity. Expression of the epsA gene promoter was higher in the ΔcomQ mutant than in the wt strain, indicating better biofilm formation. The lower activity of the DegS-DegU system in the ΔdegQ mutant affects colony morphology, while the dynamics of colony formation and their final size are similar to those of the wt strain. The promoters of proH and epsA genes are expressed later and less intensely, indicating poorer adaptation to increased salinity and inhibited biofilm formation. The highest expression of srfAA gene promoter was in the ΔdegQ mutant, followed by the wt and ΔcomQ mutant. From the results obtained, we can conclude that the absence of the ComQXPA system accelerates the adaptation of the ΔcomQ mutant to increased salinity, while the lower activity of the DegS-DegU system slows down the adaptation of the ΔdegQ mutant to increased salinity. |
