Natural competence for transformation is an important driver of horizontal DNA exchange between different organisms. This can result in accumulation of dangerous genetic features, such as antibiotic resistance genes, in a single organism. One example of an organism that frequently acquires antibiotic resistance genes is Acinetobacter baumannii, a Gram-negative opportunistic pathogen that has recently become problematic in hospital settings. Even though natural competence for transformation was demonstrated for some A. baumannii isolates, its competence regulon, the exact mechanism of DNA uptake, and the contribution of transformation to the acquisition of antibiotic resistance genes were largely unexplored at the beginning of this thesis. The aim of this thesis was therefore to better characterize optimal conditions of competence, the underlying competence regulon, and the necessary set of machinery proteins. Additionally, we aimed to identify the limiting factors that oppose transformation-mediated exchange of DNA in a small selection of strains. Here, we first addressed the general aspects of natural transformation, such as the competence window and the DNA uptake machinery. Our results showed that transformation is growth phase-dependent in A. baumannii and correlated with type IV pili (T4P) production. We demonstrated that T4P are essential for transformation and surface-associated motility but are only produced in a subfraction of the bacterial population. Furthermore, T4P production and assembly were under control of the PilSR two component system and the chemotaxis-like Pil-Chp system, respectively. These two regulatory systems were essential for competence development in A. baumannii, which is in contrast to what was observed for its close relative A. baylyi. We also investigated the reasons for non-transformability of certain A. baumannii isolates. We showed that comM interruption as well as the diversity of PilA protein sequences did not explain the strains' non-transformability. Instead, decreased expression of certain pilus genes was associated to the absence of transformability, which was also reflected in the protein level of the major component of T4P - PilA. Since the previously identified regulators could not explain the reduced transcript levels of the respective pilus genes, a transposon sequencing screen was performed to identify novel transformation-relevant genes. By comparison of transcriptional profiles of such genes between the transformable and non-transformable strains, we identified possible candidates that might explain non-transformability. Lastly, we explored whether the epigenome can influence A. baumannii's transformability. Our results showed that the source of transforming DNA has a significant effect on transformation of certain, yet not all tested strains. Specifically, for strain A118 the transformation levels were significantly decreased when non-self DNA was used as the donor DNA. Consequently, we identified a A118-specific restriction modification (RM) system that methylated specific DNA sequences in this strain and fostered the discrimination of self versus foreign DNA. Collectively, the findings of this thesis deciphered several important aspects of natural transformation in A. baumannii, which might ultimately help to better understand the spread of antibiotic resistance genes in this organism.