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    Validating Three Anti-Phage Defense Systems in Pseudomonas putida
    (Tartu Ülikool, 2025) Polekauskaite, Ilze; Ainelo, Andres, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Bacteriophages (phages) are viruses that infect bacteria, causing significant mortality. Due to this selective pressure, bacteria contain anti-phage defense systems, proteins which help to abrogate or reduce the effects of infection. As biotechnology employs genetic editing techniques to optimize microbial strains, understanding their genome is necessary to guide these decisions. Aside from efficient performance or production, anti-phage defense systems are an important consideration for minimizing risks to bacterial survival. The soil bacterium Pseudomonas putida prominently figures in synthetic biology research due to its favourable characteristics. Despite this, the anti-phage defense systems of P. putida, and the biotechnological PaW85 strain, are not studied extensively due to the lack of isolated phages. In this study, we use the CEPEST P. putida phage collection to verify three predicted anti-phage defense systems, PD-T7-1, HerA/DUF4297 and RMII. Mutant strains lacking the respective systems were constructed and phage susceptibility assays were conducted to test the function of each system in anti-phage defense. Ultimately, the roles of PD-T7-1 and RMII could not be verified with the phages available, while HerA/DUF4297 was seen to have defensive function. Further, using a strain with an active site mutation in DUF4297 showed that the enzyme likely functions as a nuclease in the complex. In the future, the expansion of the CEPEST collection will enable to further test the potential anti-phage defense roles of PD-T7-1 and RMII. Additionally, the validation of defensive function initiates future research in characterizing the activation mechanisms of HerA/DUF4297 in P. putida. Together, the findings expand the current body of knowledge of the anti-phage defense systems in P. putida and may improve future biotechnology applications.