Biology subjects

Curtis, B. A.

Publications and source records attributed to Curtis, B. A..

3 recordsLinked to original sources

bioRxiv2024

The fate of artificial transgenes in Acanthamoeba castellanii

In this study, artificial transformation experiments were performed to investigate how the A. castellanii genome responds to foreign DNA presented in both circular and linear plasmid form. Nanopore sequencing was used as a high throughput method to screen for transgene DNA in the resulting transformant cultures, and candidate transgene integrations were identified. Molecular biology experiments were performed to validate the sequence data and provide additional context on the fate of transgenes. A method was devised to estimate the rate of read chimerism in nanopore sequencing runs and accurately account for the effects of read chimerism in identifying putative transgene integrations. Based on the experimental data in hand, a potential mechanism for transgene maintenance in A. castellanii is proposed, one in which incoming foreign DNA is tandemly duplicated and telomeres are added to the ends. This nascent linear molecule is maintained as a transgene-bearing minichromosome, while also allowing for chromosomal integration.

molecular biology↗

bioRxiv2023

A unique symbiosome in an anaerobic single-celled eukaryote

Symbiotic relationships drive evolutionary change and are important sources of novelty. Here we demonstrate a highly structured syntrophic symbiosis between species of the anaerobic protist Anaeramoeba (Anaeramoebae, Metamonada) and bacterial ectosymbionts. We dissected this symbiosis with long-read metagenomics, transcriptomics of host and symbiont cells coupled with fluorescent in situ hybridization (FISH), and microscopy. Genome sequencing, phylogenomic analyses and FISH show that the symbionts belong to the Desulfobacteraceae and were acquired independently in two different Anaeramoeba species. We show that ectosymbionts likely reside deep within cell surface invaginations in a symbiosomal membrane network that is tightly associated with cytoplasmic hydrogenosomes. Metabolic reconstructions based on the genomes and transcriptomes of the symbionts suggest a highly evolved syntrophic interaction. Host hydrogenosomes likely produce hydrogen, acetate, and propionate that are consumed by the symbionts dissimilatory sulfate reduction, Wood-Ljungdahl and methylmalonyl pathways, respectively. Because the host genome sequences encode several vitamin B12-dependent enzymes but appear to lack the ability to biosynthesize this vitamin, we hypothesize that the symbionts supply their hosts with B12. We detected numerous lateral gene transfers from diverse bacteria to Anaeramoeba, including genes involved in oxygen defense and anaerobic metabolism. Gene families encoding membrane-trafficking components that regulate the phagosomal maturation machinery are notably expanded in Anaeramoeba spp. and may be involved in organizing and/or stabilizing the symbiosomal membrane system. Overall, the Anaeramoebae have evolved a dynamic symbiosome comprised of a vacuolar system that facilitates positioning and maintenance of sulfate-reducing bacterial ectosymbionts.

evolutionary biology↗

bioRxiv2021

A free-living protist that lacks canonical eukaryotic DNA replication and segregation systems

Cells must replicate and segregate their DNA with precision. In eukaryotes, these processes are part of a regulated cell-cycle that begins at S-phase with the replication of DNA and ends after M-phase. Previous studies showed that these processes were present in the last eukaryotic common ancestor and the core parts of their molecular systems are conserved across eukaryotic diversity. However, some unicellular parasites, such as the metamonad Giardia intestinalis, have secondarily lost components of the DNA processing and segregation apparatuses. To clarify the evolutionary history of these systems in these unusual eukaryotes, we generated a high-quality draft genome assembly for the free-living metamonad Carpediemonas membranifera and carried out a comparative genomics analysis. We found that parasitic and free-living metamonads harbor a conspicuously incomplete set of canonical proteins for processing and segregating DNA. Unexpectedly, Carpediemonas species are further streamlined, lacking the origin recognition complex, Cdc6 and other replisome components, most structural kinetochore subunits including the Ndc80 complex, as well as several canonical cell-cycle checkpoint proteins. Carpediemonas is the first eukaryote known to have lost this large suite of conserved complexes, suggesting that it has a highly unusual cell cycle and that unlike any other known eukaryote, it must rely on novel or alternative set of mechanisms to carry out these fundamental processes.

evolutionary biology↗