Comparative Genomic Analysis of Acanthamoeba Endosymbionts Highlights the Role of Amoebae as a "Melting Pot" Shaping the Rickettsiales Evolution.

Amoebae have been considered as a genetic "melting pot" for its symbionts, facilitating genetic exchanges of the bacteria that co-inhabit the same host. To test the "melting pot" hypothesis, we analyzed six genomes of amoeba endosymbionts within Rickettsiales, four of which belong to Holosporaceae family and two to Candidatus Midichloriaceae. For the first time, we identified plasmids in obligate amoeba endosymbionts, which suggests conjugation as a potential mechanism for lateral gene transfers (LGTs) that underpin the "melting pot" hypothesis. We found strong evidence of recent LGTs between the Rickettsiales amoeba endosymbionts, suggesting that the LGTs are continuous and ongoing. In addition, comparative genomic and phylogenomic analyses revealed pervasive and recurrent LGTs between Rickettsiales and distantly related amoeba-associated bacteria throughout the Rickettsiales evolution. Many of these exchanged genes are important for amoeba-symbiont interactions, including genes in transport system, antibiotic resistance, stress response, and bacterial virulence, suggesting that LGTs have played important roles in the adaptation of endosymbionts to their intracellular habitats. Surprisingly, we found little evidence of LGTs between amoebae and their bacterial endosymbionts. Our study strongly supports the "melting pot" hypothesis and highlights the role of amoebae in shaping the Rickettsiales evolution.