Studies on the genome biology and evolution of Acanthamoeba

Abstract

Acanthamoeba castellanii has the potential to be a model organism for studying genome evolution and lateral gene transfer in eukaryotes. However, its complex and dynamic genome has posed a challenge to realizing the full potential of this system. To lay a foundation for developing A. castellanii as an experimental model, and to gain insight into eukaryote genome biology as a whole, I undertook a multi-pronged investigation. Generating high-quality reference genome sequences of A. castellanii strains Neff and C3 revealed inter-strain variation in gene content that conferred different functions to each strain. These chromosome-scale assemblies also allowed genome structure and organization to be predicted, demonstrating an inferred karyotype of 35 chromosomes for both strains, ranging from 100 Kbp to 2.5 Mbp in size. Artificial transformation experiments were performed to investigate how the A. castellanii genome responds to foreign DNA. Using nanopore sequencing and molecular biology experiments, a potential mechanism for transgene maintenance was elucidated, where incoming foreign DNA is tandemly duplicated and telomeres are added to the ends. This nascent linear molecule is maintained as a minichromosome bearing the transgenes, while also allowing for chromosomal integration. A large amount of long- and short-read sequence data were generated from genome assembly and analysis of transformation. These sequence read sets were used to better understand the ploidy in A. castellanii. Aneuploidy was suggested by the data, with significant variation in ploidy signal across chromosomes within an isolate, as well as across isolates. The predicted proteomes generated for the new A. castellanii Neff and C3 genomes were used to conduct a phylogenomic analysis for lateral gene transfer in these two strains. This analysis revealed a trend toward lateral gene transfers that may increase metabolic flexibility, and demonstrated how A. castellanii may be taking advantage of the genetic capabilities from organisms in its community to thrive in the environment. Collectively, these findings provide a deeper understanding of Acanthamoeba genome biology, and suggest genome biology across eukaryotes may be more dynamic than previously thought.