Life Sciences Commons^™

Use Of Transposon Screening For Salicylic Acid-Assisted Desiccation Killing In Salmonella, Shannon D. Elliott

Electronic Theses and Dissertations

Salmonella enterica serovar Typhimurium is one of the most prevalent food-borne pathogens, affecting millions around the world every year, making it a threat to global health. Salmonella possesses the ability to survive the normally lethal condition of desiccation, however, discovery of the genes and mechanisms behind this phenomenon are still ongoing. Using a transposon mutagenesis approach to construct a broad transposon library, this study aimed to uncover genes that may be contributing to changes in Salmonella’s survivability under desiccation, particularly when exposed to the antimicrobial molecule salicylic acid. Building on previous findings showing salicylic acid can alter cell viability …

The Stringent Response In Pseudomonas Aeruginosa Influences The Phenotypes Controlled By The Gac/Rsm System, Michael Shawn Hooker

Electronic Theses and Dissertations

Pseudomonas aeruginosa is a ubiquitous, opportunistic pathogen that causes acute and chronic infections. Infection is typically initiated via motile and virulent strains. After exposure to stressors, acute infections make both genotypic and phenotypic switches to a chronic, sessile strain. This is due to intricate regulatory networks directing gene expression in response to stressors. One network, GacA/GacS, has been established to control virulence factors. The stringent response of bacteria is mediated by alarmones produced primarily by RelA which responds to starvation.

To study the effect of the stringent response on the virulence switch. A series of experiments were run in both …

Determining The Effects Of Maternal Adiposity On Preterm Neonatal Microbiome And Short Chain Fatty Acid Profiles, Dalton James, William A. Clark Phd, Kristy L. Thomas

Undergraduate Honors Theses

The gut microbiota and its metabolites have vast impacts on the human digestive system, immune system, and health outcomes. Short chain volatile fatty acids (SCVFAs) present in feces can be representative of the interactions of the microbiota present in the gut. Low microbiota diversity in the human gut is highly associated with obesity and adverse health outcomes. Furthermore, the maternal microbiome has a direct impact on neonatal microbiota through various pathways such as environment, skin flora, breast milk composition, and vaginal secretions. This study is aimed to further understand the associations between various factors (maternal adiposity, gestational time, length of …

Cyclic Di-Gmp Regulates Motility, Biofilm Formation, And Desiccation Tolerance In Acinetobacter Baumannii, Garrett Reynolds

Electronic Theses and Dissertations

Acinetobacter baumannii is an increasingly multidrug-resistant pathogen contributing to hospital-acquired infections necessitating the discovery of novel treatments. A bacterial second messenger, cyclic diguanosine monophosphate (cyclic di-GMP), can regulate various persistence factors that are potentially advantageous for survival in hospital environments. Cyclic di-GMP–modulating enzymes and cyclic di-GMP–binding effectors predictively are encoded in the Acinetobacter baumannii genome. I hypothesized that cyclic di-GMP controls motility, biofilm formation, and desiccation tolerance in Acinetobacter baumannii. Disrupting cyclic di-GMP–modulating enzymes or cyclic di-GMP–binding effectors should alter the regulatory effectiveness of these phenotypes. I tested the multidrug-resistant isolate Acinetobacter baumannii strain AB5075 and identified several transposon …

Characterizing The Interaction Between Candida Albicans And Two Enterobacter Species, Abigail Cornett

Electronic Theses and Dissertations

Candida albicans is the most common human fungal pathogen. The relationship between C. albicans and Enterobacter bacteria have yet to be explored. The hypothesis of this study is that C. albicans and both E. aerogenes and E. cloacae have a positive relationship and work together to infect the host. In this study, the physical cell-to-cell interaction, molecular components of said interaction, and the impact of the interaction on a live organism were explored. Results indicate that Enterobacter adheres to C. albicans and inhibits growth with unidentified secreted molecules. Als1p has potential involvement in the attachment of E. cloacae to C. …

Partial Expression Of The Vbss Gene In Rhizobium Leguminosarum Atcc 14479 And In-Silico Analysis Of The Vbs Gene Cluster In Various Microorganisms, Afreen Siddiqui

Undergraduate Honors Theses

Iron is extremely important for many organisms. Despite its abundancy, it exists in insoluble forms that makes its usability difficult. Some organisms secrete siderophores, low molecular weight compounds, that can chelate iron and convert it into usable forms for cells. One such organism, Rhizobium leguminosarum, is a nitrogen fixing symbiont proteobacteria that infects leguminous plants. The genome of Rhizobium leguminosarum ATCC 14479, which infects the red clover, Trifoli pratense, has previously been completely sequenced in our lab. Our lab has identified several genes in this strain involved in the biosynthesis of a siderophore, vicibactin. The protein product of one of …

Synergistic Inhibition Of Resistant Enterobacteriaceae Using A Possible Klebsiella Secreted Bacteriocin With Broad-Spectrum Antibiotic, Andrew Robbins

Undergraduate Honors Theses

Due to the increasing prevalence of multi-drug resistant (MDR) bacteria, it is now important to begin the search for novel means of defending against such resistant infections. Enterobacteriaceae is a clinically relevant family of bacteria that has shown extensive resistance to many antibiotics, especially after biofilm formation. Inhibitory poly-microbial interactions within this family have been observed. It is known that Citrobacter freundii (CF) growth is significantly inhibited by Klebsiella pneumoniae (KP) through a secreted protein. In this study, the potential KP bacteriocin was screened for its inhibitory effects on CF at various phases of biofilm development. The suspected KP bacteriocin …

Characterization Of The Broad-Spectrum Inhibitory Capability Of Alcaligenes Faecalis And A. Viscolactis Against Potential Pathogenic Microorganisms, Andrew Fuqua

Undergraduate Honors Theses

The recent rise of multidrug resistant microorganisms has grown from an isolated concern to a massive public health crisis. It has become imperative that scientists look for new ways to combat this issue. Due to the selective pressures of competition, bacteria and other microbes possess a host of defenses and weapons designed to exploit vulnerabilities in other microorganisms. Consequently, the study of these systems and microbial interactions has much to reveal in the search for novel antimicrobial treatments. Previous research from our laboratory has discovered that both Alcaligenes faecalis and Alcaligenes viscolactis, two rarely studied and generally non-virulent bacteria, …

The Effects Of Farnesol, A Quorum Sensing Molecule From Candida Albicans, On Alcaligenes Faecalis, Savannah Hutson

Undergraduate Honors Theses

Quorum sensing molecules have become a recent focus of study to learn if and how they can be used, both on their own and in conjecture with current antimicrobial methods, as a means of bacterial control. One such quorum sensing molecule is the sesquiterpene alcohol, Farnesol, which is synthesized and released by the fungus, Candida albicans. In most in-vivo cases, our laboratory has shown that Alcaligenes faecalis overtakes C. albicans, preventing its growth. However, as a way to counteract this inhibitory effect, Farnesol may be one way that Candida has found to fight back. In this study, we …

A Study Of The Polymicrobial Inhibitory Interactions Between Alcaligenes Faecalis And Staphylococcus Aureus, Blakeley Griffin

Undergraduate Honors Theses

Members of the Staphylococcus genus are found as a part of normal microflora in humans and can commonly be found on the skin or in the nasal cavity. However, these microorganisms can cause serious and life-threatening opportunistic infections when there is a break in the physical barrier of skin. These infections have become difficult to treat as resistant strains emerge, particularly Methicillin Resistant Staphylococcus aureus (MRSA). MRSA has become a commonly acquired nosocomial infection which is difficult to treat with conventional antibiotics of the blactam class. Even Vancomycin, a last resort antibiotic, has been ineffective on some infections. Furthermore, …

Second Messenger Cyclic-Di-Gmp Regulation In Acinetobacter Baumannii, Justin Deal

Undergraduate Honors Theses

Over time, “superbugs,” or bacteria that have become resistant to antibiotics, have become a great concern in modern medicine. Viable alternates are currently being looked into as effective and safe ways to prevent or treat infections caused by these superbugs. One such method is through the utilization of the second messenger molecule cyclic-di-GMP (c-di-GMP) that has been shown to regulate phenotypes within other bacteria that may control surface colonization in Acinetobacter baumannii. Through a series of experiments, the active enzymes that create c-di-GMP - diguanylate cyclases - and break down c-di- GMP - phosphodiesterases - have been inactivated in …

The Chelation Of Metal Ions By Vicibactin, A Siderophore Produced By Rhizobium Leguminosarum Atcc 14479, Joshua Stinnett

Undergraduate Honors Theses

Vicibactin is a small, high-affinity iron chelator produced by Rhizobium leguminosarum ATCC 14479. Previous work has shown that vicibactin is produced and secreted from the cell to sequester ferric iron from the environment during iron-deplete conditions. This ferric iron is then transported into the cell to be converted into ferrous iron. This study uses UV-Vis spectroscopy as well as ion trap-time of flight mass spectroscopy to determine that vicibactin does form a complex with copper(II) ions, however, at a much lower affinity than for iron(III). Stability tests have shown that the copper(II)-vicibactin complex is stable over time. The results of …

Determination Of The Effects That A Previously Uncharacterized Secreted Product From Klebsiella Pneumoniae Has On Citrobacter Freundii And Enterobacter Cloacae Biofilms, Cody M. Hastings

Undergraduate Honors Theses

More so than ever, Multiple Drug Resistant (MDR) bacteria are on the rise due to overuse of antibiotics along with natural selection for adaptations that enhance drug-resistant properties. One particular bacterial family, Enterobacteriaceae, has been problematic, exhibiting several bacterial members that have developed a precipitous resistance to modern antibiotics and are also primary causative agents of nosocomial, or hospital acquired, infections. Citrobacter freundii (CF) and Enterobacter cloacae (ECL) are two species of the Enterobacteriaceae family causing significant medical concern due to their role in producing numerous opportunistic infections such as bacteremia, lower respiratory tract infections, urinary tract infections, and endocarditis. …

Cloning And Expression Of C-Terminal Fragment Of Tonb From Rhizobium Leguminosarum Atcc 14479, Brooke E. Baxter

Undergraduate Honors Theses

The TonB-ExbB-ExbD complex is essential for the siderophore mediated acquisition of iron by Gram negative bacteria. The system provides energy from the proton motive force to the outer membrane in order for the iron siderophore complex to enter into the cell. The main protein involved in energy transduction, TonB, has been extensively studied in the species Escherichia Coli. It has been determined that the protein consists of 239 amino acids. In comparison, however, the TonB of Rhizobium leguminosarum consists of 457 amino acids with the same conserved regions. What is in question, therefore, is how the additional amino acids …

Effects Of Lactobacillus Rhamnosus Milk Isolate On The Production Of Inflammatory Cytokines In Enterocytes, Beverly C. Ngeny

Electronic Theses and Dissertations

In the gastrointestinal tract, probiotics have been shown to promote host immunity and to regulate immune signaling pathways. This study used Caco-2 cell line to examine the effects of a Lactobacillus rhamnosus isolate from “amabere amaruranu” a Kenyan traditional cultured milk, on the production inflammatory cytokines in enterocytes. Live Lactobacillus rhamnosus (MRS6AN), its cytoplasmic fraction (CF), filtered spent broth (FSB) or heat inactivated FSB (HIB) were used as treatments on differentiated Caco-2 cell monolayer in transwells. Cytokine content in the cell lysates, apical and basolateral supernatants were determined using ELISA. Caco-2 cell lysate treatments showed significantly increased anti-inflammatory TGF-β (ng/ml) …

Identification Of Genes Required To Synthesize An Antibiotic-Like Compound From The Soil Bacterium Rhodococcus Sp. Mtm3w5.2, Amber L. Ward

Electronic Theses and Dissertations

Rhodococcus is a soil bacterium, member of the Actinobacteria, and a close relative of the prolific small molecule producer Streptomyces. Recent interest in Rhodococcus as an under investigated source of possible bioactive secondary metabolites is sparked by the discovery of many polyketide synthase and non-ribosomal peptide synthetase genes of unknown function from sequenced Rhodococcus genomes. Rhodococcus species strain MTM3W5.2 was recently shown to produce a strong inhibitory compound with activity against most strains of Rhodococcus and closely related genera. A goal of this investigation is to discover the gene(s) required to synthesize this inhibitory molecule. The engineered Rhodococcus …

Algr Directly Controls Rsma In Pseudomonas Aeruginosa, Tyler Speaks

Electronic Theses and Dissertations

Pseudomonas aeruginosa is a bacterial pathogen that can infect any human tissue. The lungs of cystic fibrosis patients become chronically infected with Pseudomonas aeruginosa. Virulence factor gene expression is under elaborate regulatory control that remains poorly characterized. Understanding the regulatory hierarchy involved during infection is essential for identifying novel drug targets. RsmA is a post-transcriptional regulatory protein that controls expression of several virulence factors. Previous studies demonstrated alginate regulatory components AlgU and AlgR as regulators of rsmA expression. The aim of this study was to determine how AlgR controls rsmA expression. Western blot analysis of HA-tagged RsmA confirmed lower …

Characterization Of Putative Exbb And Exbd Leads To The Identification Of A Potential Tol-Pal System In Rhizobium Leguminosarum Atcc 14479, Valeria Barisic

Electronic Theses and Dissertations

Rhizobium leguminosarum is a Gram negative nitrogen-fixing soil bacterium. Due to the limited bioavailability of iron, bacteria utilize siderophores that scavenge and bind available iron. The transport of iron-siderophore complexes is achieved by the TonB-ExbB-ExbD complex. We have previously shown that a functional TonB protein is necessary for iron transport by creating ΔtonB mutants and assessing their growth and 55Fe-siderophore transport ability. We attempted to identify and characterize the roles of putative exbB and exbD genes using a similar approach. Growth curves and sequence analyses suggest putative exbB and exbD may be the tolpal-associated genes tolQ and tolR. Phenotypic and …

Influence Of The Anti-Hiv Drug Elvitegravir On Chlamydial Development And The Characterization Of Chlamydial Polymorphic Membrane Protein Expression In Herpes Simplex Virus (Hsv)/C. Trachomatis Co-Infected Cells, Hena Yakoob

Undergraduate Honors Theses

Chlamydia trachomatis is the most common bacterial agent of sexually transmitted infections worldwide and a common co-infection in AIDS patients. Chlamydial genital tract infections are often asymptomatic; therefore many infections go untreated and result in complications like chronic inflammation, ectopic pregnancy, and pelvic inflammatory disease. Chlamydia share a unique developmental cycle and under stress, can enter a state known as persistence, in which the bacteria are noninfectious but still viable. Removal of the stressor allows the chlamydiae to re-enter and complete the developmental cycle. Exposure to low-dose quinolones can cause the chlamydiae to enter persistence and halt the developmental cycle. …

Investigating Potential Bioactive Compounds From Rhodococcus And Their Effects On Mcf7 Breast Cancer Cells, Megan N. Crabtree

Electronic Theses and Dissertations

Many drugs used in the treatment of various cancers are derived from or influenced by compounds from nature. The soil bacterium Rhodococcus is of interest because of its identified secondary metabolic pathways and the production of novel natural antibiotics from several strains. In this study, a solid agar extraction method was used to collect compounds from strains of Rhodococcus. These bacterial compound extracts were then tested using a MTT assay in order to evaluate their effectiveness in augmenting MCF7 breast cancer cell death. The results of two way ANOVA analyses revealed 18 compound extracts from 15 strains of Rhodococcus that …

An Investigation Of Bacterial Ribonucleases As An Antibiotic Target, Ashley Denise Frazier

Electronic Theses and Dissertations

Antibiotics have been commonly used in medical practice for over 40 years. However, the misuse and overuse of current antibiotics is thought to be the primary cause for the increase in antibiotic resistance.

Many current antibiotics target the bacterial ribosome. Antibiotics such as aminoglycosides and macrolides specifically target the 30S or 50S subunits to inhibit bacterial growth. During the assembly of the bacterial ribosome, ribosomal RNA of the 30S and 50S ribosomal subunits is processed by bacterial ribonucleases (RNases). RNases are also involved in the degradation and turnover of this RNA during times of stress, such as the presence of …

Isolation Of A Rhodococcus Soil Bacterium That Produces A Strong Antibacterial Compound., Ralitsa Bogomilova Borisova

Electronic Theses and Dissertations

Rhodococci are notable for their ability to degrade a variety of natural and xenobiotic compounds. Recently, interest in Rhodococcus has increased due to the discovery of a large number of genes for secondary metabolism. Only a few secondary metabolites have been characterized from the rhodococci (including 3 recently described antibiotics). Twenty-four new Rhodococcus strains were isolated from soils in East Tennessee using acetonitrile enrichment culturing and identified using 16S rRNA analysis. Forty-seven Rhodococcus strains were screened for antibiotic production using a growth inhibition assay. One strain, MTM3W5.2, had 90% similarity to the Rhodococcus opacus 16S rRNA gene sequence and produced …

Requirement Of Ssdelseed-Motif Of Escherichia Coli F1FO Atp Synthase In Antimicrobial Peptide Binding., Junior Kom Tayou

Electronic Theses and Dissertations

F₁F_O ATP synthase is a membrane bound enzyme capable of synthesizing and hydrolyzing ATP. Lately, α-helical cationic peptides such as melittin and melittin related peptide (MRP) were shown to inhibit E. coli ATP synthase. The proposed but unconfirmed site of inhibition is βDELSEED-motif formed by the residues 380-386, located at the interface of α/β subunit of ATP synthase. This project was a mutagenic analysis of βDELSEED-motif residues to understand the binding mechanism and mode of action of peptide inhibitors. The study addressed 2 main questions: Are the antibacterial/anticancer effects of these peptides related to their inhibitory action …

Discovery And Characterization Of An Antibiotic From The Soil Bacterium Bacillus Sp., Thomas S. Barber

Electronic Theses and Dissertations

Many important antibiotics have become nearly obsolete due to the rise of antibiotic resistant pathogens. Rhodococcus, an actinomycete related to the prolific antibiotic producing genus Streptomyces, harbors over 30 genes for secondary metabolism that could be involved in antibiotic production. Several antibiotics have already been reported for Rhodococcus, suggesting the genus may be a good source for new inhibitory compounds. Fifty four soil bacteria were isolated using enrichment culture techniques (including 37 Rhodococcus) and screened for antibiotic producers. BTHX2, a species of Bacillus was found to have activity against Micrococcus luteus and Rhodococcus erythropolis. BTHX2 …

Modulation Of Alpha-Subunit Visit-Dg Sequence Residues Ser-347, Gly-351 And Thr-349 In The Catalytic Sites Of Escherichia Coli Atp Synthase., Laura Elaine Brudecki

Electronic Theses and Dissertations

Binding of inorganic phosphate (P_i) in ATP synthase catalytic sites is a crucial step for the synthesis of adenosine-5'-triphosphate (ATP). ATP is the fundamental means of cellular energy in almost every organism, and in order to gain insight into the regulation of ATP catalysis, critical amino acid residues responsible for binding P_i must be identified. Here, we investigate the role of highly conserved α-subunit VISIT-DG sequence residues αSer-347, αGly-351, and αThr-349 in P_i binding. Mutations αS347A/Q, αG351Q, αT349A/D/R, βR182A, and αT349R/βR182A were generated via site directed mutagenesis. Results from biochemical assays showed that αSer-347 is required …

Isolation Of A Siderophore Produced By Methicillin-Resistant Staphylococcus Aureus Strain H372., Rachel Elizabeth Presswood

Electronic Theses and Dissertations

Iron is necessary for many cellular processes such as the electron transport chain and gene regulation. However, most iron on earth is found in insoluble iron-hydroxide complexes. In addition, iron is tightly sequestered in the human body by proteins such as transferrin, making it unavailable for pathogens. In order to overcome these limitations bacteria have evolved siderophores. Siderophores are low molecular weight compounds that bind ferric iron with a high affinity. Staphylococcus aureus is an important human pathogen that is known to produce at least four siderophores, and these siderophores contribute to its virulence. S. aureus strain H372 was found …

Inhibition Of Escherichia Coli Atp Synthase By Polyphenols And Their Derivatives., Prasanna Keerthi Dadi

Electronic Theses and Dissertations

We have studied the inhibitory effect of natural and structurally modified polyphenols on Escherichia coli ATP synthase to test (I) if the beneficial dietary effects of polyphenols are related to their inhibitory actions on ATP synthase, (II) if inhibitory effects of polyphenolic compound could be augmented through structural modifications, and (III) if they can act as antimicrobial agent through their actions on ATP synthesis. X-ray crystal structures of polyphenol binding sites suggested that polyphenols bind at a distinct polyphenol binding pocket, at the interface of α,β,γ-subunits. We found that both natural and modified polyphenols inhibit E. coli ATP synthase to …

Isolation And Identification Of The Siderophore "Vicibactin" Produced By Rhizobium Leguminosarum Atcc 14479., William H. Wright Iv

Electronic Theses and Dissertations

Siderophores are small, iron chelating molecules produced by many bacteria to help meet the iron requirements of the cell. Multiple metabolic functions require iron as it serves as a cofactor in many enzymes and cellular processes. However, in the presence of oxygen and at physiologic pH, iron forms insoluble ferric complexes that cause the nutrient to be unavailable to bacterial cells. Siderophores alleviate this limitation by chelating the ferric iron, rendering it soluble and available for uptake. One group of microorganisms known for their ability to produce siderophores is the rhizobia. These bacteria are characterized both by their formation of …

Molecular Mechanism Of Ferricsiderophore Transport Via The Outer Membrane Receptor Fhua In Escherichia Coli., Jennifer K. Cooke

Electronic Theses and Dissertations

Iron is essential for life and growth in most organisms. Although it is abundant, iron exists mostly as insoluble iron-oxyhydroxide. Bacteria secrete siderophores to chelate iron and transport it into the cell via specific outer membrane receptors. The FhuA receptor protein transports ferrichrome, a siderophore produced by Ustilago sphaerogena. We determined the binding affinity of variants from the conserved 'lock region' of FhuA and also created and characterized variants of the highly conserved R452 to determine its role in ferrichrome transport. We hypothesize that during transport the plug domain of FhuA does not leave the barrel; rather it undergoes …

Iron Acquisition In Rhodococcus Erythropolis Strain Igts8: Characterization Of A Mutant Strain That Over Produces Siderophore., Melanie Anne Pratt

Electronic Theses and Dissertations

Iron is an essential nutrient for most bacteria because enzymes like nitrate reductase and cytochromes use it as a cofactor. However, in most aerobic, neutral pH environments, iron is essentially insoluble and not easily available for bacteria to use. Many bacteria respond to this problem by releasing small organic compounds called siderophores that bind and effectively solubilize iron so that it can be transported into the cell for growth. The focus of this study was to learn more about the iron acquisition and especially the transport of iron by the soil bacterium Rhodococcus erythropolis. To fulfill this aim, mutant …