Life Sciences Commons^™

The Algz/R Two-Component System Is Responsible For Attenuation Of Virulence In Pseudomonas Aeruginosa, Danielle A. Williams

Electronic Theses and Dissertations

Pseudomonas aeruginosa is an important opportunistic pathogen. Many P. aeruginosa virulence factors are regulated by the AlgZ/R two component system. AlgZ is the sensor histidine kinase which phosphorylates AlgR, the response regulator. AlgR activates transcription of different gene targets based upon its phosphorylation state. The genes that encode AlgZ and AlgR are transcribed in an operon. While regulation of algR expression has been well studied, regulation of algZ expression has not. Using a pilW mutant in concert with algZTF-lacZ transcriptional fusion, we conducted a transposon mutagenesis to identify algZ regulators. We identified an unknown autoregulatory loop. The type …

Range-Wide Prevalence And Impacts Of Pseudocercosporella Inconspicua On Lilium Grayi And An Assessment Of L. Superbum And L. Michauxii As Reservoirs, Cindy L. Barrett

Electronic Theses and Dissertations

Lilium grayi (Gray’s Lily), a southern Appalachian endemic species, is threatened by a Lilium-specific fungal pathogen, Pseudocercosporella inconspicua. The disease is characterized by tan lesions that can cause early senescence, while also lowering seed production and viability. This project tested for P. inconspicua conidia and accessed health at nine locations. The disease was present and ubiquitous across the range of L. grayi. Through identification of P. inconspicua conidia in the field, L. superbum (Turk’s Cap Lily) was identified as an additional host, while L. michauxii (Michaux’s Lily) was disease-free. However, infection was inducible in both species. With …

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 …

Toll Like Receptor 4 Stimulation Increases Scavenger Receptor A Expression On Murine Macrophages, Mackenzie L. Guthrie

Undergraduate Honors Theses

Sepsis is the body’s response to an overwhelming infection and is a serious consequence of critical illness. It can cause tissue damage, organ failure, and death. Sepsis continues to have an unacceptably high mortality rate, due to the lack of effective treatments. Specific therapeutic targets for sepsis remain elusive since the complex functional changes that result in a septic state remain poorly understood. Macrophage Scavenger Receptor A (SRA, CD204) is a surface receptor that binds negatively charged, endogenous and exogenous ligands. We have discovered that SRA plays a significant role in the pathophysiology of sepsis. We have shown that mice …

Comparison Of Anti-Pneumococcal Functions Of Native And Modified Forms Of C-Reactive Protein, Donald Neba Ngwa

Electronic Theses and Dissertations

The anti-pneumococcal function of native C-reactive protein (CRP) involves its binding to phosphocholine molecules present on Streptococcus pneumoniae and subsequent activation of the complement system. However, when pneumococci recruit complement inhibitory protein factor H on their surface, they escape complement attack. Non-native forms of CRP have been shown to bind immobilized factor H. Accordingly, we hypothesized that modified CRP would bind to factor H on pneumococci, masking its complement inhibitory activity, allowing native CRP to exert its anti-pneumococcal function. As reported previously, native CRP protected mice from lethal pneumococcal infection when injected 30 minutes before infection but not when injected …

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) …

Influence Of Lactobacillus Rhamnosus Isolated From “Amabere Amaruranu” Cultured Milk On Adipogenesis, Justin E. Kotala

Electronic Theses and Dissertations

This study was performed to test the in vitro effects of a Lactobacillus rhamnosus isolate from “amabere amaruranu”, a traditional Kenyan cultured milk, on 3T3-L1 and Caco-2 cell lines. Cultures of fully mature 3T3-L1 adipocytes were treated with bacterial isolate cell extract (CE), filtered spent broth (FSB) from overnight bacterial culture, or with a PBS control. Expression levels of PPAR³1 and 2, C/EBP±, and ATGL proteins in 3T3-L1 cells were upregulated by FSB treatment. CE treatment did not affect protein expression levels. Expression of MTTP and SREBP-1c proteins in Caco-2 cells showed no change with either treatment. Optical density measurements …

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 …

Identification Of Transcription Factors Gzf3, Rfx1, Orf19.3928 As Being Implicated In Candida-Bacterial Interactions., Joni Watson

Undergraduate Honors Theses

Candida albicans is an opportunistic pathogen that is present in the normal flora in a majority of individuals. One key factor in C. albicans virulence is the ability to change its morphology from yeast to an elongated or hyphal form. The regulation of this morphogenesis relies in part upon quorum sensing (QS) molecules. C. albicans often exists as part of a mixed culture alongside other microbes and is influenced by their presence as well as the presence of QS molecules that they produce. In this study, a library of diploid homozygous transcriptional regulator knockout (TRKO) mutants were screened to identify …

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. …

Probiotic Potential Of Bacterial Isolates From ‘Amabere Amaruranu’ Cultured Milk, Blaise B. Boyiri

Electronic Theses and Dissertations

Probiotics are viable nonpathogenic microbes that positively affect host health. Probiotics inhibit infection, activate immunity, and promote mucosal-barrier development. Many microbes have probiotic activity. Nonetheless, the selection of stable strains and their specific mechanism(s) of action are not fully elucidated. Bacteria from ‘Amabere amaruranu’ cultured milk from Kenya were isolated and identified by PCR sequence analysis of the 16S rRNA gene. Isolates were examined for stability to acid and bile, antimicrobial activity, mucin production, and degradation and sensitivity to antibiotics, hence their potential for probiotics. Lactobacillus isolates were acid unstable, bile-stable, nonmucinolytic, and presented antibacterial activity. L. rhamnosus cell fractions …

Polymorphic Membrane Protein Expression In Chlamydia/Hsv Co-Infected Cells, Julia S. Colgrove

Undergraduate Honors Theses

The Chlamydiaceae are a bacterial family that contains a single genus: Chlamydia. The genus Chlamydia consists of 9 species that are obligate, intracellular pathogens. Untreated C. trachomatis infections can lead to serious health ramifications, such as ectopic pregnancy, tubal factor infertility, pelvic inflammatory disease, and long-term pelvic pain. In this study, it was found that a primary antibody dilution of 1:400 using methanol fixed HeLA cells, as derived from Carrasco, et al. protocol, was only optimal for PMP-C staining. Pmp-A, Pmp-B, and Pmp-F were found to stain brighter with formaldehyde fixed, infected HeLa cells and using different primary antibody dilutions. …

Characterization Of Tonb In Rhizobium Leguminosarum Atcc 14479, Brian D. Hill

Electronic Theses and Dissertations

Rhizobium leguminosarum is a gram-negative soil bacterium that requires iron for survival. However, iron becomes insoluble in the presence of oxygen at physiological pH. In response, Rhizobia species have used siderophore mediated iron transport systems to meet their iron requirements. R. leguminosarum ATCC 14479 produces the trihydroxymate siderophore vicibactin and we hypothesize that the import of the ferric iron-vicibactin complex is energized by the TonB-ExbB-ExbD system. Here, we have identified a putative tonB gene. A tonB mutant was created and compared with wild type in its ability to transport ⁵⁵Fe-vicibactin. Also, the putative TonB of R. leguminosarum ATCC …

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 …

Identification And Characterization Of Genetic Factors Involved In Candida-Bacterial Interactions, Sean J. Fox

Electronic Theses and Dissertations

Throughout existence, fungi and bacteria have long shared ecological niches and thus engage in numerous interactions to mutually enhance survival or antagonistically gain competitive advantages. Of importance to human health are those interactions that involve bacteria with the opportunistic fungi, Candida albicans. An important virulence factor of C. albicans is the ability to control morphology, which allows the transition between yeast, pseudohyphal, and hyphal phenotypes. Morphological control in C. albicans is governed by quorum sensing and the secreted autoregulatory molecule farnesol. Quorum sensing allows individual cells to sense the environment and respond as a group. Bacteria also use quorum …

Proximity-Labeling Of Near Neighbors Of Lamin A And Lamin A-Δ50 (Progerin)., Mohammad Sabri

Undergraduate Honors Theses

In an attempt to isolate and identify proteins that differentially interact with or locate near lamin A and progerin, we used a previously described method named BioID (proximity-dependent biotin identification). This method is based on fusion of a promiscuous E. coli biotin-protein ligase (BL) to a targeting protein (in this study, lamin A-GFP and progerin-GFP). The biotin ligase biotinylates amino residues in proteins that are near-neighbors of the fusion protein. To create the fusion proteins, BL was sub-cloned from a pcDNA3.1 MCS-BirA(R118G)-HA plasmid donated by Kyle Roux from University of South Dakota. The BL fragment was ligated into a pNEBR-X1-lamin …

Detection Of Bacterial Retroelements Using Genomics, Sen Mu

Electronic Theses and Dissertations

The reverse flow of genetic information can occur when a special DNA polymerase called Reverse Transcriptase (RT) copies the genetic information in an RNA molecule back into a complementary DNA. One type of RT encoding gene found in bacteria is called a retron element. Recent bacterial genome sequencing projects have revealed many examples of retron RT genes. This gene assignment is based on comparison with a few known retron RT proteins. However, RT proteins are highly diverse in their amino acid sequences, and thus the assigned identity of these RT proteins as retrons in genome databases is questionable. One way …

A Mathematical Model For Antibiotic Resistance In A Hospital Setting With A Varying Population, Edward H. Snyder

Electronic Theses and Dissertations

Antibiotic-resistant bacteria(ARB) is causing increased health risk and cost to society. Mathematical models have been developed to study the transmission of resistant bacteria and the efficacy of preventive measures to slow its spread within a hospital setting. The majority of these models have assumed a constant total hospital population with the admission and discharge rates being equal throughout the duration. But a typical hospital population varies from day to day and season to season. In this thesis, we apply variable admission and discharge daily rates to existing deterministic and stochastic models which examine the transmission of single and dual resistant …

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 …