The Rickettsiae comprise intracellular bacterial symbionts and pathogens infecting diverse eukaryotes. Here, we provide a detailed characterization of 'Candidatus Jidaibacter acanthamoeba', a rickettsial symbiont of Acanthamoeba. The bacterium establishes the infection in its amoeba host within 2 h where it replicates within vacuoles. Higher bacterial loads and accelerated spread of infection at elevated temperatures were observed. The infection had a negative impact on host growth rate, although no increased levels of host cell lysis were seen. Phylogenomic analysis identified this bacterium as member of the Midichloriaceae. Its 2.4 Mb genome represents the largest among Rickettsiales and is characterized by a moderate degree of pseudogenization and a high coding density. We found an unusually large number of genes encoding proteins with eukaryotic-like domains such as ankyrins, leucine-rich repeats and tetratricopeptide repeats, which likely function in host interaction. There are a total of three divergent, independently acquired type IV secretion systems, and 35 flagellar genes representing the most complete set found in an obligate intracellular Alphaproteobacterium. The deeply branching phylogenetic position of 'Candidatus Jidaibacter acanthamoeba' together with its ancient features place it closely to the rickettsial ancestor and helps to better understand the transition from a free-living to an intracellular lifestyle.

Highlights d Mediator kinases CDK8 and CDK19 are distinct transcription regulators in the IFN-g pathway d CDK8 acts as a kinase while CDK19 functions as a scaffold in driving IFN-g gene profiles d CDK8 and CDK19 activate distinct gene sets d Mediator kinase CDK8 promotes polymerase II pause release during the IFN-g response

Amoebae serve as hosts for various intracellular bacteria, including human pathogens. These microbes are able to overcome amoebal defense mechanisms and successfully establish a niche for replication, which is usually the cytoplasm. Here, we report on the discovery of a bacterial symbiont that is located inside the nucleus of its Hartmannella sp. host. This symbiont, tentatively named ‘Candidatus Nucleicultrix amoebiphila', is only moderately related to known bacteria (∼90% 16S and 23S rRNA sequence similarity) and member of a novel clade of protist symbionts affiliated with the Rickettsiales and Rhodospirillales. Screening of 16S rRNA amplicon data sets revealed a broad distribution of these bacteria in freshwater and soil habitats. ‘Candidatus Nucleicultrix amoebiphila' traffics within 6 h post infection to the host nucleus. Maximum infection levels are reached after 96–120 h, at which time point the nucleus is pronouncedly enlarged and filled with bacteria. Transmission of the symbionts occurs vertically upon host cell division but may also occur horizontally through host cell lysis. Although we observed no impact on the fitness of the original Hartmannella sp. host, the bacteria are rather lytic for Acanthamoeba castellanii. Intranuclear symbiosis is an exceptional phenomenon, and amoebae represent an ideal model system to further investigate evolution and underlying molecular mechanisms of these unique microbial associations.