Life Sciences Commons^™

Multifaceted Approach To Understanding Acinetobacter Baumannii Biofilm Formation And Drug Resistance, Jessie L. Allen

USF Tampa Graduate Theses and Dissertations

Acinetobacter baumannii is a multi-drug resistant nosocomial pathogen known for causing wound related- and respiratory-infections. It is currently on the WHO’s list of critical pathogens due to its broadly drug resistant nature and the constant appearance of pan-resistant isolates. A majority of the infections caused by this organism are biofilm associated, however there is limited existing knowledge regarding the mechanisms used to engage in this multicellular lifestyle. As such, we set out to explore the factors influencing this behavior using an 10,000+ isolate transposon mutant library of A. baumannii strain AB5075. Of the strains tested, 6.45% demonstrated some level of …

Unraveling The Role Of Novel G5 Peptidase Family Proteins In Virulence And Cell Envelope Biogenesis Of Staphylococcus Aureus, Stephanie M. Marroquin

USF Tampa Graduate Theses and Dissertations

Virulence factors and the bacterial cell envelope are two important components in S. aureus pathogenesis and survival. More importantly, understanding the regulation of these cellular processes is crucial to further understanding and combating this successful pathogen. To date, numerous factors have been identified as playing a role in the regulation of Agr activity in S. aureus, including transcription factors, antisense RNAs, and host elements. Herein we investigate the product of SAUSA300_1984 (termed MroQ), a transmembrane G5 peptidase family protein, as a novel effector of this system. Using a USA300 mroQ mutant we observed a drastic reduction in proteolysis, hemolysis, and …

Microbial Dark Matter: Culturing The Uncultured In Search Of Novel Chemotaxonomy, Sarah J. Kennedy

USF Tampa Graduate Theses and Dissertations

Although <1% of environmental isolates can be cultured, insights into the culturable fraction refine future techniques to unlock the cryptic biodiversity. This cryptic biodiversity is omnipresent within culture-independent surveys of environmental bacterial populations. Subsequently, these elusive organisms are termed “microbial dark matter” due to the intrigue surrounding their potential taxonomic and biosynthetic impacts. To this end of investigating bacterial-specific dark matter, we employed high-throughput culturing techniques to examine Gulf of Mexico sediments and sponges for chemotaxonomically important bacteria. Through stepwise implementation of permissive and selective recovery techniques, we succeeded in recovering over 150 unique bacterial isolates. We observed several isolates that pose as chemotaxonomically interesting, including several strains that belong to critical bioremediation taxa. In addition to these biotechnological-amenable isolates, we have also succeeded in culturing taxa historically attributed with antibacterial discoveries. One such isolate was a previously sponge-associated organism belonging to the rarely cultured Verrucosispora genus. Through extensive comparative genomics, we identified it as a novel species and subsequently named it Verrucosispora sioxanthis sp. nov. with the published genome serving as a resource for continued microbial dark matter investigations. Following continued passaging of the organism, we observed improved growth, biomass, and liquid growth dispersal within each increasingly sub-passaged generation of the isolate. As such, the laboratory attenuation, or domestication, of the organism warranted in-depth whole genomic analyses to generate insight into the increased laboratory sustainability. Through phenotypically-guided bioinformatics, we determined that the domestication was mediated through genome-wide selective pseudo- or non-pseudogenization of genes. As the previously sponge-associated organism evolved with the selection pressures of the static laboratory, we demonstrate that isolate effectively underwent genomic atrophy to resolve into a largely stable, sustainable laboratory-domesticated strain. Collectively, our chemotaxonomic discovery pipeline expands on the culturable bacterial biodiversity and ultimately provides a previously uncultured model organism for initial forays into resolving the microbial dark matter knowledge gap.

Investigation Of An Alternative Protocol For The Production Of Sars-Cov-2 Antigenic Proteins, Nichole Ninaltowski

USF Tampa Graduate Theses and Dissertations

With the COVID-19 pandemic showing no signs of slowing down, large-scale antigenic protein production is still needed for surveillance using serologic assays. From screening to vaccines to biotherapeutics, being able to produce the proteins for these assays is essential; however, the current gold standard method for producing SARS-CoV-2 spike proteins is prohibitively expensive for most research groups.

Alternative methods of transfecting mammalian cells to produce recombinant proteins that are relatively inexpensive have been used for years. Unlike the expensive, commercially available lipid-based methods, other established methods such as polyethyleneimine (PEI), are considerably easier, and cheaper to meet the needs of …

A Functional Characterization Of The Omega (Ω) Subunit Of Rna Polymerase In Staphylococcus Aureus, Shrushti B. Patil

USF Tampa Graduate Theses and Dissertations

In bacteria, RNA polymerase (RNAP) is a well-characterized and highly conserved multi-subunit enzyme complex responsible for transcription of DNA into RNA. The ω subunit (rpoZ/RpoZ), one of the smaller accessory subunits of RNAP, is often overlooked and under-studied, however, particularly in Gram-positive species. We have previously shown that deleting ω impacts the integrity of RNA polymerase in S. aureus, especially the β' subunit, and alters preference of the core enzyme for sigma factors, skewing heavily towards σB instead of the housekeeping σ factor. Consequently, this causes deregulation of myriad transcriptional processes, strongly rewiring gene expression circuits, and ultimately impairing the …

Transcriptomic And Functional Investigation Of Bacterial Biofilm Formation, Brooke R. Nemec

USF Tampa Graduate Theses and Dissertations

Staphylococcus aureus and Acinetobacter baumannii are two highly successful human pathogens, which have adopted very different, but effective survival strategies. The success of S. aureus is attributed to the tight regulation of an arsenal of virulence factors. Conversely, A. baumannii lacks what would be considered traditional virulence factors and, instead, has developed a high tolerance for environmental stress, which allows it to persist in unforgiving environments, including nosocomial settings and the human body. One common characteristic of these two organisms is their proclivity for biofilm formation. Herein, we discuss the diverse mechanisms governing biofilm formation for A. baumannii and S. …

Exploring Prokaryotic And Viral Communities Within Florida’S Freshwater Springs, Kema Saydoon Malki

USF Tampa Graduate Theses and Dissertations

Florida’s freshwater springs serve as a crucial ecosystem for the state, having both ecological and economic importance. Springs occur where there is an opening where aquifers discharge groundwater. Florida’s springs are fed by the Floridan aquifer system (FAS), which underlies the entire state and extends into several neighboring states. The FAS provides potable water to over 10 million people. The constant flow of groundwater through the springs feeds major river systems and creates unique freshwater systems that support numerous plants and animals. The combination of natural beauty and charismatic fauna attract tourism and generate millions of dollars in revenue for …

Development And Validation Of An Isothermal Amplification Assay For Eastern Equine Encephalitis Virus, Mikayla D. Maddison

USF Tampa Graduate Theses and Dissertations

Eastern Equine Encephalitis virus (EEEV) is a neurotrophic alphavirus for which there is no effective treatment or vaccine for humans. Periodic outbreaks in the Eastern United States represent an ongoing public health problem; Florida serves as the reservoir for EEEV for the rest of the country. Reverse transcription-polymerase chain reaction (RT-PCR) is the current gold standard for molecular diagnostic testing of the presence of EEEV in vectors. However, RT-PCR is technically complex and can be difficult for mosquito control districts to utilize. In order to provide a simple, cost-effective alternative for mosquito surveillance and control, a novel one step reverse …

Novel Co2 Transporters From Autotrophic Bacteria From Extreme Environments, Sarah E. Schmid

USF Tampa Graduate Theses and Dissertations

Autotrophic organisms are responsible for introducing carbon into food webs. These organisms take up dissolved inorganic carbon (DIC) and use it as their major carbon source, converting it to biomass using a variety of carbon fixing pathways (e.g., the Calvin-Benson-Bassham cycle). In nature, DIC concentrations can be low or temporally heterogeneous, so autotrophic organisms adapt with CO2 concentrating mechanisms (CCMs) to facilitate the acquisition and fixation of this dissolved gas. CCMs consist of membrane-spanning DIC transporters, which elevate intracellular DIC concentrations, which in turn are harvested by carboxysomes, proteinaceous microcompartments that contain the carboxylase Ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO) and carbonic anhydrase. …