Origins of cin: Lateral Gene Transfer of Cytoplasmic Incompatibility Nuclease Operon to Orientia tsutsugamushi

CinB nucleases are Wolbachia proteins that induce cytoplasmic incompatibility (CI) through tandem nuclease domains nuc1 and nuc2 1. CI is a form of reproductive parasitism (RP) whereby males are conditionally sterilized 2. The system behaves as a toxin-antidote (TA) system 1-8 where operon gene cinA encodes an antidote and gene cinB encodes a toxin 1,5. Cin operons are purportedly the cause of gene-drive induced by wolbachiae infecting Drosophila simulans 1,9-12. An unanswered research question is whether lateral transfer of CI operons to bacteria outside wolbachiae would transfer RP phenotype and activate gene-drive. We demonstrate that a cin operon, capable of gene-drive, has jumped into the genome of Orientia tsutsugamushi, a human pathogen and causative agent of lethal scrub typhus. When expressed in transgenic Drosophila melanogaster, the wildtype cinBoTsu was capable of inducing CI independent of its partner antidote cinAoTsu. In addition, cinAoTsu rescued the phenotype in accordance with strict TA functionality. To understand the diverging roles of the tandem nuclease domains we mutated the domains and tested all permutations of active/inactive forms. Finally, we isolated IS5 transposon variants flanking the operon in O. tsutsugamushi and re-activated them to test their mobility. We demonstrate that these transposons can transfer genes and initiate lateral gene transfers into E. coli. These data demonstrate that active bacterial transposons can mobilize and transfer CI factors (cifs) to diverse bacteria. Overall, our data contribute mechanistic understanding in support of the TA model of CI and illuminate biochemical mechanisms that mobilize cifs across genomes from phylogenetically diverse taxa. Significance StatementCI operons are foundational genes that directly contribute to the success of Wolbachia-based bio-control strategies. Two applications of RP-inducing Wolbachia strains are insect population replacement and the incompatible insect technique. Both these techniques do not work if cifs do not function. Thus, a mechanistic understanding of cif function contributes to worldwide bio-control implementations. Furthermore, certain wolbachiae have long been studied as broad-spectrum inducers of diverse RP phenotypes, including CI, parthenogenesis, male-killing, and feminization. How these diverse phenotypes evolve and if they are all induced by cif genes is a long-standing field question. For example, Orientia tsutsugamushi, the causative agent of a deadly scrub typhus, induces parthenogenesis in Leptotrombidium mites, which putatively have reproductive advantages over uninfected mites. How Orientia induces RP is an important epidemiological question in vector biology. In our report, we show that an active cin operon is capable of CI and jumped into Orientia genomes via an IS5 transposon. We reconstructed this transposon and engineered it as a biotechnological tool. In toto, our study leads to the proposal of a new hypothesis whereby CI and parthenogenesis phenotypes might both be connected to the same cif expressed under divergent host genetic contexts.