Several studies have been conducted on the epidemiology of the virulent pathogen X. fastidiosa subsp. pauca strain ‘De Donno’ that is causing a severe disease in olive trees, but limited data are available on its phenotypic and biological traits. To fill this knowledge gap, the in vitro behaviour of the strain was investigated, exploring relevant biological features, like growth rate, biofilm formation, cell-cell aggregation, and twitching motility, making comparison with the recognised type strain Temecula1, belonging to subsp. fastidiosa. The strain ‘De Donno’ had enhanced biofilm production and showed a more aggregative phenotype than the strain ‘Temecula 1’. Specific strains of X. fastidiosa have been shown to have natural competence, a biological trait that is related to its ability to uptake the DNA from the external environment. Using the established protocols and in spite of several attempts with different DNA sources the strain ‘De Donno’ failed to show natural competence. This lack of natural competence is consistent with the Citrus-infecting pauca strains. Genetic manipulation was possible with electroporation and mutants were successfully obtained in the presence of an inhibitor of the Type I restriction-modification (T1RM) system. Two different plasmids containing the chromosomal replication origin (oriC) of X. fastidiosa and E. coli, respectively, were used to transform X. fastidiosa ‘De Donno’ strain to produce a GFP-expressing and a knockout strain for rpfF gene, an enoyl-CoA hydratase, belonging to the crotonase family enzyme, that is involved in producing a diffusible signal factor (DSF) used for Quorum sensing. The outcomes of this study shed light on peculiar traits of the strain ‘De Donno and produced functional mutants that will be useful to gain novel insights into the biology of this strain and its pathogenicity mechanisms.