22 Feb 2019

Diversity of Bacteriophages in the Genomes of Nitrogen-Fixing Bacteria

Maddie Mickelson '19

Bacteriophages are viruses that infect bacteria and are found in every habitat; however, the abundance and diversity of these viruses is poorly understood. Phages contain a high proportion of novel genetic sequences and represent the largest reservoir of unexplored genes. Examining the diversity and abundance of bacteriophages is essential to maximize their biotechnological applications and understand the evolutionary relationships between bacteria and viruses. The rhizosphere is the zone of soil which centers around plant roots, and harbors a diverse community of microbes. Nitrogen-fixing bacteria are crucial for nutrient cycling because they convert atmospheric nitrogen to ammonia, which plants incorporate into proteins and DNA. The purpose of this study is to use bioinformatics techniques to investigate the abundance and diversity of bacteriophages in nitrogen-fixing bacteria and develop an assay for the environmental detection of these phages. We hypothesize that genomes of nitrogen-fixing bacterial genera (Sinorhizobium, Bradyrhizobium, Mesorhizobium and Rhizobium) contain multiple bacteriophage sequences that can be classified into groups based on their DNA sequence similarity. To assess phage abundance, 498 nitrogen-fixing bacterial genomes were obtained from the NCBI Genome database and analyzed using PHASTER, a program which detects bacteriophage sequences. One to thirteen bacteriophage sequences were detected in the genomes of the bacteria examined. These organisms contained an average of two bacteriophage sequences per chromosome. To assess diversity, viral genomes were retrieved and analyzed using the MAFFT program. A phylogenetic tree constructed using these data revealed that the bacteriophages clustered into seven different groups. PhiSiGns was used to identify conserved viral proteins and design primers to detect phages in soil samples by the polymerase chain reaction. A primer set was developed based on a conserved viral methyltransferase. Positive DNA amplification results were obtained using various soil samples, indicating this assay could be used for detection of phages in the environment.

From Tyrosine to Coenzyme Q in Yeast

Sylvia Toledo '19

Coenzyme Q (CoQ) is a redox lipid that serves many functions in the cell. One of the most well known functions of CoQ is the transfer of electrons from complex I to complex II in the electron transport chain (ETC), helping to form the proton electrochemical gradient that drives ATP synthesis in the mitochondria. A defect in the biosynthesis of CoQ causes CoQ deficiencies, which in turn causes serious diseases such as myopathy or cerebellar ataxia. Today, CoQ deficiencies are treated through CoQ supplements, however, there have been cases where this fail due to the highly hydrophobic nature of CoQ. The biosynthesis of CoQ from the aromatic precursor 4-hydroxy-benzoic acid (4-HB) is well understood. It is also known 4-HB derives from the amino acid tyrosine, which is a common pathway in eukaryotes. However, details of the biosynthesis from tyrosine to 4-HB is not well understood. The goal of this study is to elucidate which enzymes and intermediates play a role in the conversion of tyrosine to 4-HB in the model yeast species Saccharomyces cerevisiae. Selective mutagenesis and genetic screening were used to determine genes involved in the pathway from tyrosine to 4-HB. Plasmids and genetic complementation were used to recover effects observed in mutated cells. Several intermediates and enzymes were identified, namely 4-hydroxyphenylpyruvate (4-HPP), 4-hydroxybenzaldehyde (4-Hbz) and Hfd1, an aldehyde dehydrogenase. It was found that the aromatic precursor of CoQ from tyrosine is derived via the synthesis of 4-HPP and the oxidation of 4-Hbz to 4-HB by the aldehyde dehydrogenase Hfd1. Mutation in the Hfd1 gene resulted in CoQ deficiency. Synthesis of CoQ in cells lacking Hfd1 was recovered by the expression of the human aldehyde dehydrogenase ALDH3A1, which suggest a similar pathway in humans. In future studies, it would be of interest to know if the pathway from tyrosine to 4-HB found in yeast is conserved in humans, if so, hydrophilic 4-HB can instead be used as a alternative supplement to treat CoQ deficiencies.