Date of Award
1-1-2023
Document Type
Thesis
Degree Name
M.S. in Pharmaceutical Science
First Advisor
Paul Boudreau
Second Advisor
Marc Slattery
Third Advisor
Jason Paris
School
University of Mississippi
Relational Format
dissertation/thesis
Abstract
Cyanobacteria are well-known for their production of a vast array of secondary metabolites. Cyanobacteria are found in marine, aquatic, and terrestrial environments. Due to their production of an abundance of natural products, cyanobacteria may have significant applications in biotechnological, industrial, agricultural, and pharmacological fields. Although they have many beneficial implications, they are also known producers of Harmful Algal Blooms (HABs). These HABs can exude toxins into the marine environments, while decomposition of their biomass can create hypoxic zones. These HABs can lead to ecosystem destruction, wildlife death, and human health impacts. In order to better understand the metabolic capabilities and natural product potential of these organisms it is important to have complete genomes of these organisms. Complete genome sequencing allows genome mining for biosynthetic gene clusters and an insight into these metabolic capabilities. However, cyanobacteria have multiple morphologies from colonial, filamentous, to single cellular, adding a hurdle to developing a universal DNA isolation protocol. In addition to differing morphologies, cyanobacteria also possess specialized plant-like cell walls that cause difficulty for traditional chemical lysis protocols. These cell walls often make the chemical lysis steps of commercialized bacterial DNA isolation kits inadequate for cell lysis. However, the lysis steps of commercial plant DNA isolation kits that can successfully recover DNA can also be too vigorous, leading to sheared DNA that is incompatible with long-read sequencing technologies. Therefore, our first goal in this research was to develop a protocol sufficient for cyanobacterial and algal DNA extraction with multiple morphologies, that is suitable for complete genome assemblies using in house Oxford Nanopore MinION sequencing.
One class of cyanobacterial natural products is the siderophores. Siderophores are small molecules that chelate iron to produce bioavailable iron compounds for the organism. By being able to sequester environmental iron cyanobacteria are more readily able to survive under low iron stress. The chelation of these iron compounds may have applications in heavy metal bioremediation or the pharmaceutical industry Therefore, in this research our second goal was to investigate the metabolomics of cyanobacteria under iron limitation stress. To do so, we prepared CAS (chrome azurol S) dyed-freshwater BG-11 plates to test for siderophore production, and then cultured environmentally isolated from cyanobacteria fresh water Northern Mississippi environments in iron-limited freshwater BG-11 liquid media, prioritizing the CAS-positive strains for metabolomic analysis. Once the cultures were harvested from the liquid media the supernatant was than fractionated for a run on our LCMS-QTOF. The data from this experiment were analyzed using GNPS, Global Natural Products Social Molecular Networking, and MassQL, Mass Query Language, tools to evaluate for the production of putative siderophores.
In this research, we optimized three repetitive lysis steps to enrich the ratio of cyanobacteria to heterotrophic DNA, leading to an increase in cyanobacterial DNA. The Zymo clean and concentrator column and protocol were then used for purification and SERAMAG left side size selection beads were used twice at a 1:1 ratio of beads:sample to skew the read DNA isolation towards longer molecules. From this preparation, with the use of bioinformatics tools the complete cyanobacteria genome of both BL-A-14 and BL-A-16 were assembled. The screening for siderophore production also yielded positive hits for siderophores using the MassQL tool. In future work, the DNA protocol should be used in the isolation of DNA over all 53 Boudreau Lab isolates to acquire complete genomes for our strain library. Future studies should also investigate the production of siderophores in freshwater BG-11 media, due to the presence of iron-adducts found via the MassQL in the freshwater BG-11 media as well as the iron limited medium.
Recommended Citation
Wagner, Alexis Dawn, "Genomic and Metabolomic Investigation into Xenic Cyanobacterial and Algal Cultures from Northern Mississippi" (2023). Electronic Theses and Dissertations. 2593.
https://egrove.olemiss.edu/etd/2593