Genome sequencing is a technique that identifies the order of nucleotides in a DNA sequence of an organism. This process is the most successful for large animals like humans and plants, however, genome sequencing of microbial eukaryotes is still advancing. Microbial Eukaryotes like Amoeba have very diverse and complex genomes making it more difficult to sequence than other larger organisms. The goal of this research is to collect pure DNA from amoebozoans for genome sequencing. The challenge is that amoeba live with bacteria and other small eukaryotes that make it difficult to discern amoeba DNA from others before sequencing. This relative contamination is problematic because these other organisms usually have simple genomes that are easily sequenced in comparison to amoeba with complex genomes. This complicates the process and cost of genome sequencing in amoebae. In this study, two methods are considered to isolate the cells for pure DNA extraction including antibiotic treatment and centrifugation including density gradient centrifugation and nuclear isolation. Preliminary results of the antibiotic treatment proved to be ineffective because the concentrations of the antibiotic used more detrimental to the cells of amoebae than the bacteria. DNA isolation based on centrifugation showed promising result. Development of a protocol for pure genomic DNA isolation will be useful in other research fields that depend on pure genomic DNA.

Sexual reproduction is a tool used to increase genetic diversity amongst species. Trichosphaerium, a genus of amoeba, is understudied compared to other eukaryotic microbes. This genus was described as sexual consisting of two stages, a gamont (gamete-forming) and schizont (spicule-containing) stage. The genetic basis of the sexual gamount stage has never been investigated. In this study, a genetic approach was taken to identify the presence or absence of specific genes present during the gamont stage of Trichosphaerium species. A sex-related gene inventory analysis, containing eleven meiosis-specific genes and 31 fusion genes, was conducted using a custom-made bioinformatics pipeline searching the transcriptomes of three morphotypes including small, medium, and large Trichosphaerium isolates. It was expected for medium and large amoeba to identify several sex-related genes in their transcriptomes, since the cells and nuclei are more likely to fuse when larger compared to when they are smaller. Results support the presence of several meiosis-specific genes in the transcriptomes of medium and large Trichosphaerium isolates, specifically DMC1, MND1, HOP2, MSH5, and MSH4, which are involved in crossover regulation. In conclusion, the identification of meiosis-specific genes in species part of the genus Trichosphaerium supports the claim that the gamont stage of this species is sexual.

Trichosphaerium marine amoeba are a unique case when attempting to observe the processes that occur during its life cycle. Trichosphaerium has two forms: asexual (schizont) and sexual (gamont), but there is limited literature on the phenomenons that occur during its life cycle. The stages and mechanisms of gamont forms were portrayed through detecting changes in morphology and gene expression analysis. We observed the changes in Trichosphaerium behavior throughout three organismal stages that related to amoeba size: small, medium, and large. Physical measurements of amoeba dimensions were taken as an indication of the difference in morphology of each of these groups and stages. Immunohistochemistry was performed to track the progression of development leading to plasmogamy and karyogamy within each growing amoeba to confirm the occurrence of nuclear fusion. The transcriptome of each group of amoebas within a set life cycle stage were then sequenced to perform differential expression analysis in order to quantify the changes in gene expression among these stages. This can give insight into the mechanisms that are occurring within each stage as a result of a certain level of expression, giving a better understanding of the development and reproduction of this organism.

 
Neisseria gonorrhoeae, a pathogenic sexually-transmitted bacterium, is becoming untreatable due to increased antibiotic resistance. Related commensal (non-pathogenic) Neisseria species are found in the human oral and nasopharynx, but are rarely studied. Here, we seek (1) to characterize the intrinsic antibiotic resistance (AR) profile of commensal Neisseria species: N. mucosa, N. cinerea, N. lactamica, and N. elongata; and (2) to determine whether these bacteria can be considered reservoirs of AR genes for pathogenic Neisseria. Within this work, measuring AR levels in commensal Neisseria will be conducted by Disk Diffusion Assays (DDA), Minimum Inhibitory Concentration (MIC), and Minimum Bactericidal Concentration (MBC) assays with antibiotics used to treat gonorrhea. Through these experiments, we expect commensal strains to display resistance levels similar to or greater than reference pathogenic strains. Characterizing AR profiles in commensals is necessary to understand the molecular and cellular mechanisms evolved by these bacteria to resist commonly prescribed antibiotics and to determine whether commensal Neisseria species can be considered reservoirs of AR genes. It is imperative that scientists identify the mechanisms of AR in commensal Neisseria in order to anticipate the progress of AR in pathogenic N. gonorrhoeae, and potentially N. meningitidis.