20.500.12556/RUL-163849 Exploring the role of Bacillus subtilis gene kamA controlled by a phage regulatory switch Proučevanje uravnave gena kamA bakterije Bacillus subtilis s fagnim regulatornim stikalom Bacillus subtilis is a model organism for studying Gram-positive microorganisms; its kamA gene can be interrupted by the inserted lysogenic phage phi3T, closely related to SPbeta which was shown to act as a phage regulatory switch regulating the expression of the spsM gene during sporulation. This thesis explores the possible antiviral activity of the kamA gene product based on similarity of its 3D structure with three viperins, a class of antiviral proteins found across the tree of life. This possible role was tested by performing multiple double-layer plaque assays, susceptibility assays in liquid cultures and a phage adsorption assay comparing B. subtilis strain PY79 and its ∆kamA and ∆spsM mutants. Results revealed that a deletion of kamA decreases susceptibility of B. subtilis to lytic phage Nf, rather than the hypothesised viperin-like activity of its gene product. Furthermore, deletion of the gene exhibits an effect on the rate of phage adsorption resulting in a trend distinct from cells with an intact gene, However, due to an unforeseen phenomenon affecting this experiment, we cannot say this with certainty. As kamA harbours an attachment site for phage phi3T which effectively has a deleterious effect, these findings demonstrate a way in which lysogenic phages may confer protection towards lytic phages. Gen kamA bakterije Bacillus subtilis, ki je modelni organizem za preučevanje grampozitivnih mikroorganizmov, je lahko prekinjen z vstavljenim lizogenim fagom phi3T, ki je soroden fagu Spbeta. Zanj je bilo dokazano, da deluje kot regulatorno fagno stikalo, ki uravnava izražanje gena spsM med sporulacijo. V tej magistrski nalogi smo preučili možno protivirusno aktivnost produkta gena kamA. Slednja aktivnost proteina je bila predvidena na podlagi podobnosti 3D modela strukture proteina KamA s strukturami treh protivirusnih proteinov, viperinov, ki jih lahko najdemo po vsem drevesu življenja. Morebitno protivirusno vlogo gena kamA smo preizkusili z več občutljivostnimi testi tako na ploščah kot v kulturi, ter s spremljanjem hitrosti adsorpcije litičnega faga Nf na gostitelja, in sicer tako, da smo primerjali vpliv faga na sev B. subtilis PY79 ter njegovi ∆kamA in ∆spsM mutanti. Rezultati so pokazali, da delecija gena kamA zmanjša občutljivost B. subtilis na fag Nf, vendar pa odpornosti ne daje domnevana viperinu-podobna aktivnost proteina KamA. Rezultati nakazujejo, da delecija gena kamA vpliva na hitrost adsorpcije faga Nf, ki se razlikuje od adsorpcije na celice divjega tipa. Vendar zaradi nepredvidenega pojava, ki je vplival na ta poskus tega ne moremo trditi dokončno. Ker lahko fag phi3T gen prekine z vstavitvijo svojega genoma v ta gen, te ugotovitve nakazujejo način, s katerim lizogeni fagi lahko vplivajo na svojega gostitelja in kako mu zagotovijo zaščito pred drugimi fagi. lysogenic bacteriophages phage regulatory-switch bacteriophage resistance Bacillus subtilis gene kamA lizogeni bakteriofagi fagno regulatorno stikalo odpornost na bakteriofage Bacillus subtilis gen kamA true false false [F. Kračman] Angleški jezik Slovenski jezik Magistrsko delo/naloga 2024-10-13 07:15:41 2024-10-13 07:15:49 2024-10-14 09:22:37 0000-00-00 00:00:00 2024 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 579.25:578.347 248303 211345155 44691.pdf 44691.pdf 1 A6473080E8EAE4012B68B2C907A9B726 02e2a9f4a083662110956e89e4fa9eb7e04379c3a103c506d9f7febd3b5c9755 ff75cd1b-8921-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=193798 Biotehniška fakulteta 0 0 0