During Type IV Secretion System (T4SS) mediated killing of neighboring bacterial cells by Xanthomonas citri (Xac), several effector proteins are secreted and injected into the target cell by a contact dependent mechanism. As a first objective the function of these effector proteins will be investigated. Assessing the activity of different effector deletion mutants against different bacterial species will allow discrimination between the antibacterial and regulatory effects of the individual effector proteins. Additionally, the ectopic and conditional expression of antibacterial effector proteins combined with protein interaction studies will point to their cellular targets. A second objective involves whole-genome transposon insertion strategies to retrieve all loci involved during competition of Xac with other bacterial species. A first straightforward classical transposon screen will reveal the clear-cut mutants and this search will be expanded by a Tn-seq approach, drastically increasing our genetic view on the interbacterial competition of Xac. Additionally, this objective could also reveal alternative mechanisms working either independently or together with the T4SS during interbacterial competition. As a final objective, the regulation, assembly and localization of the T4SS during contact with target cells will be further analyzed by combining time-lapse fluorescence microscopy with different fluorescent labeling approaches.
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