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Bakterije Bacillus subtilis sintetizirajo več različnih sekundarnih metabolitov, med katerimi je tudi bacilen. Sinteza nekaterih sekundarnih metabolitov je uravnavana s sistemom za zaznavanje kvoruma (QS). Da bi preverili, ali je tudi sinteza bacilena uravnavana s QS, smo pripravili seve z okvarami v sistemu QS ter poročevalsko fuzijo promotorja PpksC z zapisom za rumeni fluorescenčni protein (yfp). Metode so zajemale spremljanje intenzitete fluorescence s fluorescenčnim mikročitalcem na nivoju celotne populacije in s konfokalnim fluorescenčnim mikroskopom (CLSM) na nivoju posamezne celice. Metodi nam podata različne rezultate, saj nam mikroskopija ponudi dodaten vpogled na nivo posamezne celice. Pri sevu B. subtilis PS-216 na nivoju celotne populacije lahko potrdimo, da na izražanje bacilena vpliva sistem QS, kar ne moremo reči za sev B. subtilis NCIB3610. Seva PS-216 in NCIB3610 se razlikujeta v izražanju genov za sintezo bacilena, kar lahko potrdimo tako na nivoju posamezne celice, kot tudi na nivoju celotne populacije. Z metodo CLSM smo ugotovili, da se pri vseh sevih pojavita dve podpopulaciji – prva – številčnejša z nižjim nivojem sinteze bacilena in druga – manj številčna z višjim nivojem sinteze bacilena. Poleg tega opazimo razlike v izražanju bacilena med rastjo v različnih časovnih točkah. Med sevi PS-216 divjega tipa in sevi z okvarami v sistemu QS so prisotne minimalne razlike v izražanju genov za sintezo bacilena, medtem ko pri sevih NCIB3610 statistično značilnih razlik ni. Rezultati pri uporabi metode CLSM niso bili ponovljivi, zato bi v nadaljevanju hipoteze lahko preverili z drugimi neodvisnimi metodami (na primer pretočno citometrijo), ki bi nam z večjo gotovostjo potrdile ali ovrgle zastavljene hipoteze. Bacillus subtilis synthesizes several secondary metabolites, one of which is also bacillaene. The synthesis of some secondary metabolites is regulated by the quorum sensing system (QS). In order to check whether bacillaene synthesis is also regulated by QS, we prepared strains with defects in the QS system and a reporter fusion of the PpksC promoter with yfp. The methods included monitoring fluorescence intensity with a fluorescence microplate reader at the level of the whole population, and with a confocal fluorescence microscope (CLSM) at the level of a single cell. Microscopy offers us an insight into the single-cell level therefore, these two methods gave us different results. It can be confirmed that the bacillaene expression is affected by the QS system in PS-216, which on the other hand cannot be confirmed for the NCIB3610. Strains PS-216 and NCIB3610 differ in the expression of genes required for bacillaene synthesis, which can be confirmed at the single cell level and at the level of the whole population. Using the CLSM method, we found that two populations occur in all strains - the first - more numerous with a lower level of bacillaene synthesis, and the second - less numerous with a higher level of bacillaene synthesis. In addition, we observed differences in bacillaene expression during growth at different time points. There are minimal differences in the expression of genes required for bacillaene synthesis between wild-type PS-216 and QS-defective strains, while there are no statistically significant differences between NCIB3610 strains. The results with the CLSM method were not reproducible, so the hypotheses could be tested in the future with other independent methods (for example, flow cytometry), which would confirm or refute the proposed hypotheses with greater certainty. |
