"Despite that many human diseases are caused by bacterial pathogens, not much is known about how these pathogenic bacteria emerge. The alpha-proteobacterial order Rickettsiales comprises intracellular bacterial pathogens, including the causative agent of epidemic typhus, Rickettsia prowazekii. The overall aim of the current project is to gain insight in the evolutionary transition from free-living bacterium to (human) pathogen, using the Rickettsiales as a model system. To do so, deeply branching Rickettsiales lineages will be isolated from environmental samples and from amoeba that contain Ricketsiales-like endosymbionts. Free-living Rickettsiales species will be isolated from the Sargasso Sea using a single-cell genomics approach. In parallel, protist-associated Rickettsiales species will be isolated from amoeba. Subsequently, an attempt will be made to determine the complete genome sequence of the obtained free-living and ciliate-associated Rickettsiales species. By comparing the obtained genomic data with that of their pathogenic relatives, principle differences will be revealed that should give insight in the evolutionary transition from free-living bacterium to human pathogen. The proposed study aims at enumerating a number of pathogenicity genes that could represent excellent targets for vaccine development for treatment of infections caused by pathogenic Rickettsiales species."